title: Differential LINE-1 retrotransposition in induced pluripotent stem cells between humans and great apes

city: La Jolla

first: Christopher

last: Benner

state: California

address: 9500 Gilman Dr. MC 0640

institute: University of California, San Diego (UCSD)

summary: Understanding cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic comprehension of the evolution and diversity of our own species. Until now, preserved tissues have been the main source of most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behavior, are not amenable to genetic manipulation and do not allow translation of observed differences into phenotypical divergence. We hypothesized that induced pluripotent stem cells (iPSCs) could provide a unique biological resource to elucidate relevant phenotypical differences between human and the great apes and that those differences could have potential adaptation and speciation value. Here, we describe the generation and initial characterization of iPSCs from chimpanzees and bonobos as novel tools to explore our most recent evolution. Comparative gene expression analysis of human and NHP iPSCs revealed differences in regulation of Long Interspersed Nuclear Element (LINE-1 or L1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. We observed decreased levels of L1 restricting factors APOBEC3B (A3B)7 and PIWIL28 in NHP iPSCs which was correlated with increased human and chimpanzee L1 mobility and endogenous L1 mRNA levels. Moreover, results from manipulation of A3B and PIWIL2 levels in iPSCs suggested a causal inverse relationship between levels of these proteins and L1 activity. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPSCs in culture and may have also occurred in the germline during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have had an adaptive significance.

sample_organism:
Pan paniscus
Pan troglodytes
Homo sapiens

overall_design: polyA RNA-Seq profiling of iPS cells from human, chimpanzee, and bonobo, and small RNA-Seq profiling of human iPS cells.

first: Maria

last: Marchetto

first: Inigo

last: Narvaiza

first: Ahmet

last: Denli

first: Fred

middle: H

last: Gage


title: Kinetoplastid-specific histone variant functions are conserved in Leishmania major

status: Public on Oct 26 2013

city: SEATTLE

first: Gowthaman

last: Ramasamy

state: WASHINGTON

address: 307 WESTLAKE AVE N

department: GHBC

country: USA

institute: SEATTLE BIOMEDICAL RESEARCH INSTITUTE

type: Expression profiling by high throughput sequencing

summary: Protein-coding genes in kinetoplastid protists are transcribed from polycistronic arrays, yielding RNA precursors which are processed to form mature transcripts bearing a 5’ spliced leader (SL) and 3’ poly(A) tract. Regions of transcription initiation and termination lack known eukaryotic promoter and terminator elements, and current data suggest that transcription is instead regulated predominantly through epigenetic mechanisms. Several epigenetic marks, including histone modifications,  histone variants, and an atypical DNA modification known as base J have been localized to regions of transcription initiation or termination in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Despite this conservation, the phenotypes of base J mutants vary significantly across trypanosomatids, suggesting that the specific epigenetic networks governing transcription initiation and termination have diverged significantly during evolution. In this light, we sought to characterize and compare the roles of the histone variants H2A.Z, H2B.V, and H3.V in L. major. As in T. brucei, the histone variants H2A.Z and H2B.V were shown to be essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast and again similar to T. brucei, H3.V is not essential in Leishmania as H3.V-null lines grew normally, resembled WT, and remained infectious. Using SL-primed RNA-seq, we found that H3.V-null parasites have steady-state transcript levels comparable to WT parasites and display no defects in the efficiency of transcription termination at convergent strand switch regions (SSRs). Our results show a conservation of histone variant phenotypes between L. major and T. brucei, in contrast to the phenotypes associated with the epigenetic DNA base J modification.

overall_design: Total RNA from Four LmjF samples were analyzed using RNA-Seq. One of them is wildtype parasites, one is single knockout for H3V gene and two independent double knockouts for H3V gene.

first: Britta

middle: A

last: Anderson

first: Iris

middle: L

last: Wong

first: Loren

last: Baugh

first: Peter

middle: J

last: Myler

first: Stephen

middle: M

last: Beverley


title: Systems-level analysis of N-starvation induced modifications of carbon metabolism in a Chlamydomonas starchless mutant

status: Public on Oct 24 2013

city: Los Angeles

first: Sabeeha

last: Merchant

address: 607 Charles E. Young Drive East

department: Department of Chemistry and Biochemistry

institute: University of California Los Angeles

summary: To understand the molecular basis underlying increased triacylglycerol accumulation in starchless Chlamydomonas mutants, we undertook comparative time course transcriptomics of sta6 (CC-4348), a cw strain purported to represent the parental STA6 strain (CC-4349), and 3 independent STA6 strains (STA6-C2, STA6-C4 and STA6-C6, generated by complementation of sta6, CC-4565, –4566 and –4567) in the context of nitrogen deprivation. Despite the N starvation induced dramatic remodeling of the transcriptome, there are relatively few differences (5 x 102) between sta6 and STA6, the most dramatic of which are in the abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically ICL1, MAS1, TAL1, FBP1 and PCK1, suggestive of increased carbon movement towards hexosephosphate in sta6 by up-regulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicates increased succinate and malate, increased Glc-6-P and decreased fructose-1,6-bisphosphate, which documents the impact of these changes. Comparisons of the independent datasets in multiple strains allowed the delineation of a sequence of events in a global N-starvation response in Chlamydomonas, starting within minutes with up-regulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual up-regulation of genes encoding enzymes of TAG synthesis. Finally, genome re-sequencing analysis indicated that i) the deletion in sta6 extends into the neighboring gene, RBO1, and ii) a commonly-used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype.

overall_design: The transcriptomes of five different Chlamydomonas strains (wild type CC-4349, sta6 mutant (CC-4348),and three indepdent STA6 strains (STA6-C2, STA6-C4 and STA6-C6) are profiled by RNA-Seq in timecourse experiments after the transition to minus nitrogen

first: Ian

last: Blaby

first: Anne

last: Glaesener

first: Tabea

last: Mettler

first: Sorel

last: Fitz-Gibbon

first: Sean

last: Gallaher

first: Bensheng

last: Liu

first: Nanette

last: Boyle

first: Janette

last: Kropat

first: Mark

last: Stitt

first: Shannon

last: Johnson

first: Christoph

last: Benning

first: Matteo

last: Pellegrini

first: David

last: Casero


title: Genome-wide map of p53 binding sites by ChIP-seq in  human lymphoblastoid cell lines  treated with ionizing radiation

status: Public on Oct 23 2013

city: Research Triangle Park

first: Xuting

last: Wang

state: NC

address: 111 TW Alexander Dr

institute: NIEHS/NIH

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We have used chromatin immune-precipitation with parallel sequencing (ChIP-Seq) technology to identify genome-wide p53 binding in human lymphoblastoid cell lines treated withionizing radiation

overall_design: ChIP-Seq analysis of p53 binding sites in human lymphoblastoid cells treated with ionizing radiation or vehicle

first: Michelle

middle: R

last: Campbell

first: Douglas

last: Bell


title: Genome-wide map of p53 binding sites by ChIP-seq in  human lymphoblastoid cell lines  treated with doxorubicin

summary: We have used chromatin immune-precipitation with parallel sequencing (ChIP-Seq) technology to identify genome-wide p53 binding in human lymphoblastoid cell lines treated with a DNA-damaging chemotherapeutic reagent doxorubicin.

overall_design: ChIP-Seq analysis of p53 binding sites in human lymphoblastoid cells treated with Doxorubicin or vehicle


title: Transcriptome along the murine developing gut

city: Lausanne

first: Marion

last: LELEU

address: EPFL-SV-ISREC-UPDUB- Station 19

department: School of Life Sciences

country: Switzerland

institute: EPFL

summary: Hox genes are required for the development of the intestinal caecum, a major organ of species eating plants. We have analysed the transcriptional regulation of Hoxd genes in caecal buds and show that they are controlled by a series of enhancers located in a gene desert telomeric to the HoxD cluster. The start site of two neighboring and opposite long non-coding RNAs, Hotdog and Twin of Hotdog, specifically transcribed in the caecum, contacts the expressed Hoxd genes in the framework of a topological domain, a large domain of interactions, which ensures a robust transcription of these genes during caecum budding. We show that hedgehogs have kept this regulatory potential despite the absence of caecum, suggesting that these enhancers are used in other developmental situations. In this context, we discuss some striking similarities between the caecum and the limb buds, suggesting the implementation of a common budding tool-kit.

overall_design: Transcriptional activity at the HoxD locus in the murine developing gut at E13, Differential gene expression analysis along the murine developing gut

first: S

last: Delpretti

first: D

last: Duboule


title: Genome-wide incorporation dynamics reveal distinct categories of turnover for the histone variant H3.3

city: Bethesda

first: Wenfei

last: Jin

state: MD

address: 9000 Rockville Pike

department: Systems Biology Center

institute: NHLBI,NIH

summary: We developed a system to study the DNA replication-independent turnover nucleosomes containing the histone variant H3.3 in mammalian cells. By measuring the genome-wide incorporation of H3.3 at different time points following epitope-tagged H3.3 expression, we find three categories of H3.3-nucleosome turnover in vivo: rapid turnover, intermediate turnover and, specifically at telomeres, slow turnover. Our data indicate that H3.3-containing nucleosomes at enhancers and promoters undergo a rapid turnover that is associated with active histone modification marks including H3K4me1, H3K4me3, H3K9ac, H3K27ac and the histone variant H2A.Z. The rate of turnover is negatively correlated with H3K27me3 at regulatory regions and with H3K36me3 at gene bodies.

overall_design: Examination of incorporation dynamics of histone variant H3.3

first: Daniel

middle: C

last: Kraushaar

first: Alika

last: Maunakea

first: Misook

last: Ha

first: Keji

last: Zhao


title: Transcriptome along the hedgehog developing gut

overall_design: Transcriptional activity at the HoxD locus in the hedgehog developing gut at E13.5, Differential gene expression analysis along the hedgehog developing gut


title: Transcriptome in developing caeca

summary: Hox genes are required for the development of the intestinal caecum, a major organ of species eating plants. We have analysed the transcriptional regulation of Hoxd genes in caecal buds and show that they are controlled by a series of enhancers located in a gene desert telomeric to the HoxD cluster. The start site of two neighboring and opposite long non-coding RNAs, Hotdog and Twin of Hotdog, specifically transcribed in the caecum, contacts the expressed Hoxd genes in the framework of a topological domain, a large domain of interactions, which ensures a robust transcription of these genes during caecum budding. We show that hedgehogs have kept this regulatory potential despite the absence of caecum, suggesting that these enhancers are used in other developmental situations. In this context, we discuss some striking similarities between the caecum and the limb buds, suggesting the implementation of a common budding tool-kit. Transcriptional activity at the HoxD locus in developing caeca at E13.5


title: HoxD locus

type: Genome binding/occupancy profiling by genome tiling array

type: Expression profiling by genome tiling array

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

sample_organism:
Atelerix albiventris
Mus musculus

platform_organism:
Atelerix albiventris
Mus musculus

overall_design: Refer to individual Series


title: Competition between pre-mRNAs for a limiting splicing machinery drives global changes in splicing

status: Public on Oct 22 2013

city: Santa Cruz

first: Lily

last: Shiue

state: CA

address: 1156 High Street

department: MCD Biology

institute: UCSC

summary: During meiosis in yeast, global splicing efficiency increases. The mechanism for this is relief of competition for the splicing machinery by repression of intron-containing ribosomal protein genes (RPGs). Repression of RPGs with rapamycin also increases splicing efficiency in vegetative cells. Reducing levels of an RPG-dedicated transcription factor globally improves splicing and suppresses the temperature-sensitive growth defect of a spliceosome mutation. These results indicate that the spliceosome is limiting and pre-mRNAs compete with each other. Under these conditions, splicing efficiency of a given pre-mRNA therefore depends on both its concentration and affinity for the limiting splicing factor(s) as well as those of the competing pre-mRNAs. We propose that trans-competition control of splicing helps repress meiotic gene expression in vegetative cells, and promotes efficient meiosis. Competition between RNAs for a limiting factor may be a general condition important for function of a variety of post-transcriptional control mechanisms.

overall_design: Splicing and gene expression profiles of 1) wild type yeast cells treated with rapamycin (2 biological replicates) relative to untreated cells and 2)  prp4-1 pGAL-IFH1 (down-regulated expression of IFH1 transcription factor(specific for ribosomal protein genes)) relative to prp4-1 yeast.

first: Elizabeth

last: Munding

first: Manuel

last: Ares


title: Spatial organization of chromatin at the HoxD locus in developing caeca (4C-seq)

overall_design: Chromosome Conformation Capture (4C seq) at the HoxD locus in developing caeca at E13.5


title: Comparison of microRNA Profiling Platforms (HTS)

city: Toronto

first: Shirley

last: Tam

state: Ontario

address: 661 University Avenue, Suite 510, MaRS Building, West Tower

department: Genome Technologies

country: Canada

institute: Ontario Institute for Cancer Research

type: Non-coding RNA profiling by high throughput sequencing

summary: Global miRNA expression profiling of human malignancies is gaining popularity in both basic and clinically driven research. But to date, the majority of such analyses have used microarrays and quantitative real-time PCR.  With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal, many more options. To make effective use of these different platforms, the strengths and pitfalls of several miRNA profiling technologies were assessed, including a microarray platform, NGS technologies and the NanoString nCounter System.  These results were compared to gold-standard quantitative real-time PCR.

overall_design: Comparison of non-small cell lung cancer cell lines grown in vitro (n = 5) and in vivo (n = 5) as xenograft models.

first: Richard

last: de Borja

first: Ming-Sound

last: Tsao

first: John

middle: D

last: McPherson


title: Comparison of microRNA Profiling Platforms


title: Induction of sarcomas by mutant IDH2

status: Public on Oct 21 2013

city: New York

first: Craig

middle: B

last: Thompson

state: NY

address: 1275 York Ave

department: Cancer Biology and Genetics

institute: Memorial Sloan-Kettering Cancer Center

type: Methylation profiling by high throughput sequencing

summary: More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal 20 progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that neomorphic IDH2 mutations can be oncogenic in mesenchymal cells..

overall_design: RRBS sequencing of (1) IDH wild type and mutant human chondrosarcomas, (2) isogenic cell lines expressing wild-type or mutant IDH.

first: Chao

last: Lu

first: Sriram

last: Venneti

first: Altuna

last: Akalin

first: Fang

last: Fang

first: Patrick

middle: S

last: Ward

first: Raymond

middle: G

last: DeMatteo

first: Andrew

last: Intlekofer

first: Chong

last: Chen

first: Jiangbin

last: Ye

first: Meera

last: Hameed

first: Khedoudja

last: Nafa

first: Narasimhan

middle: P

last: Agaram

first: Justin

last: Cross

first: Raya

last: Khanin

first: Christopher

middle: E

last: Mason

last: Healey

first: Scott

middle: W

last: Lowe

first: Gary

middle: K

last: Schwartz

first: Ari

last: Melnick


title: Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs: Phytophthora infestans small RNA

city: Saint Louis

first: James

last: Carrington

state: MO

address: 975 North Warson Road

institute: Donald Danforth Plant Science Center

summary: Deep sequencing of small RNAs from three Phytophthora species, P. infestans, P. ramorum and P. sojae, was done to systematically analyze small RNA-generating components of Phytophthora genomes. We found that each species produces two distinct small RNA populations that are predominantly 21- or 25-nucleotides long. We present evidence that 25-nucleotide small RNAs are short-interfering RNAs that silence repetitive genetic elements. In contrast, 21-nucleotide small RNAs are associated with inverted repeats, including a novel microRNA family, and may function at the post-transcriptional level.

overall_design: Phytophthora infestans mycelium small RNAs were sequenced and aligned to the P. infestans genome for analysis.

first: Noah

last: Fahlgren

first: Stephanie

last: Bollmann

first: Kristin

last: Kasschau

first: Josh

middle: T

last: Cuperus

first: Caroline

last: Press

last: Sullivan

first: Elisabeth

last: Chapman

first: J

last: Hoyer

first: Kerrigan

last: Gilbert

first: Niklaus

last: Grünwald


title: Mot1 redistributes TBP from TATA-containing to TATA-less promoters

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary: The Swi2/Snf2-family ATPase Mot1 displaces TBP from DNA in vitro, but the global relationship between Mot1 and TBP in vivo has been unclear.  We therefore mapped the distribution of Mot1 and TBP on native chromatin at base-pair resolution.  Mot1 and TBP binding sites coincide throughout the genome, and depletion of TBP results in a global decrease in Mot1 binding.  Using midpoint-versus-length mapping to assess the spatial relationship of Mot1 and TBP on chromatin, we find evidence that Mot1 approaches TBP from the upstream direction, consistent with its in vitro mode of action.  Strikingly, inactivation of Mot1 leads to both increases and decreases in TBP-genome association.  Sites of TBP gain tend to contain robust TATA boxes, while sites of TBP loss contain poly(dA:dT) tracts that may contribute to nucleosome exclusion.  We propose that the action of Mot1 is required to clear TBP from intrinsically preferred (TATA-containing) binding sites, ensuring sufficient soluble TBP to bind intrinsically disfavored (TATA-less) sites.

sample_organism:
Saccharomyces cerevisiae
Drosophila melanogaster

overall_design: We have analyzed the genomic distributions of yeast TBP and Mot1 using Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin and sequencing (ORGANIC-seq).

first: Gabriel

last: Zentner

first: Steven


title: Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs

sample_organism:
Phytophthora infestans
Phytophthora sojae
Phytophthora ramorum

first: Christopher


title: Transcriptome-wide interaction mapping of hnRNP L in human T cells with CLIP-seq

status: Public on Oct 20 2013

city: Philadelphia

first: Kristen

last: Lynch

state: Pennsylvania

address: 906 Stellar Chance Labs 422 Curie Blvd

department: Biochemistry and Molecular Biophysics

institute: University of Pennsylvania

summary: CLIP-seq of hnRNP L in human T cells

overall_design: Triplicate resting and stimulated human samples from both primary CD4+ and cultured JSL1 (Jurkat-derived monoclonal) were subjected to CLIP-seq to comprehensively map hnRNP L-RNA interaction sites.

first: Brian

last: Cole

first: Ganesh

last: Shankarling


title: Tmem88a Mediates GATA-dependent Specification of Cardiomyocyte Progenitors by Restricting Canonical WNT Signaling

first: Todd

last: Evans

state: New York

address: 1300 York Avenue  Box 282

institute: Weill Medical College of Cornell University

summary: We sequenced mRNA from zebrafish wild-type embryos, gata5 morphants, gata6 morphants, and gata5/6 morphants at bud and 6-somite developmental stages to identify genes co-operatively regulated by gata5 and gata6 during cardiomyocyte progenitor specification.

overall_design: Samples were collected in duplicate, with 40 embryos per sample. Single 36-base pair reads were sequenced on the Illumina Genome Analyzer IIx

first: Natasha

last: Novikov


title: Expression evolution in gene groups: Inferring non-neutral regulatory change in pathways from transcriptional profiling data

city: San Francisco

first: Yulia

last: Mostovoy

address: 555 Mission Bay Blvd South

institute: University of California, San Francisco

summary: We profiled the transcriptomes of four Saccharomyces species, as well as pairwise hybrids between three of the species with S. cerevisiae

sample_organism: Saccharomyces cerevisiae x Saccharomyces paradoxus

sample_organism: Saccharomyces cerevisiae x Saccharomyces bayanus

sample_organism: Saccharomyces cerevisiae x Saccharomyces mikatae

overall_design: For pairwise comparisons between Saccharomyces cerevisiae and each of S. paradoxus, S. mikatae, and S. bayanus, we performed 3'-end RNA-seq on RNA from each parent species and each interspecific hybrid.

first: Tiffany

middle: Y

last: Hsu

first: Rachel

last: Brem


title: Altered Epigenetic Regulation of Homeobox Genes in Human Oral Squamous Cell Carcinoma Cells

status: Public on Oct 19 2013

first: Katarzyna

middle: Maria

last: Marcinkiewicz

address: 1300 York Ave. Room E405

department: Pharmacology

institute: Weill Cornell Graduate School of Medical Sciences

summary: To gain insight into the molecular changes during OSCC carcinogenesis, we performed unbiased, whole genome deep sequencing (RNA-seq) using RNA isolated from cultured, human TERT-immortalized, non-tumorigenic OKF6-TERT1R and OSCC SCC-9 cells.

overall_design: OKF6-TERT1R cells and SCC-9 cells were  plated in 10 cm2 tissue culture plates at the density of 2 × 106 cells/plate and treated with 1 μM RA or vehicle (0.1% ethanol) for 48 hours. Experiment includes 3 independent biological replicates.

first: Lorraine

last: Gudas


title: RNA sequencing of SHP -/- knockout mouse transcriptome

city: Durham

first: Integrative

last: Bioinformatics

address: 111 TW Alexander Drive

department: BCBB

institute: NIEHS

summary: Goal of this study is to identify annotated and non-annotated genes transcriptionally regulated by small heterodime partner (SHP, Nrob2) expression.

overall_design: Liver 5' capped RNA samples from three SHP -/- and three wild type mice were sequenced with Illumina GAII sequencer.

first: Li

last: Wang

first: Curt

last: Hagedorn


title: Systematic discovery of spleen miRNAs involved in host response to avian pathogenic Escherichia coli (APEC) by deep sequencing and integrated analysis

city: Guangzhou

first: Xinzheng

last: Jia

state: Guangdong

address: Wushan Road

department: Animal Science

country: China

institute: South China Agricultural University

summary: Avian pathogenic Escherichia coli (APEC) is considered one of the most common infectious bacterial diseases resulting in significant economic losses in poultry industry worldwide. In order to investigate the association between host immune resistance and miRNA expression in the pathogenic process induced by APEC, miRNA expression profiles in broilers spleen were performed by Solexa deep sequencing from three different treatment groups including non-challenged (NC), challenged-mild pathology (MD), and challenged-severe pathology (SV).In total, 3 462 706, 3 586 689, and 3 591 027 clean reads were obtained for NC, MD, and SV, respectively. After comparing the miRNA expression patterns, 27 differentially expressed miRNAs were identified among the three response groups, which included 13 miRNAs between NC and MD, 17 between NC and SV, and 14 between MD and SV. For these miRNAs, different expression in MD and SV suggested they may have resistance activity in APEC infection. Through integrated analysis of miRNA and mRNA expression patterns, 43 negative pairs between miRNA and mRNA (r < -0.80) were obtained. 4 miRNAs were validated to be significant negatively correlated to targets by quantitative real time PCR: gga-miR-21 (CLEC3B and GGTLA1), gga-miR-429 (TMEFF2, CDC20, SHISA2 and NOX4), gga-miR-146b (LAT2 and WNK1), and gga-miR-215 (C7 and ASL2). Additionally, the expression of gga-miR-21 and gga-miR-146b was significantly up-regulated by LPS induced in HD11 macrophage cell. In contrast, gga-miR-429 has no significant change. In summary, we present the first report that characterized the miRNA profiles of chicken spleen in response to APEC infection, and identified several candidate miRNAs which might accelerate host immune response through down-regulating their specific target genes.

overall_design: Through the intra-air sac route into the left thoracic air sac, 240 non-vaccinated males at 4 weeks of age were challenged with 0.1 ml APEC O1 (10^8 colony forming units) and another 120 non-vaccinated males were non-challenged but treated with 0.1 ml PBS. Detailed information on the APEC O1 strain and challenge process was described by previously described study. Necropsy was performed at 1 day post challenge, and a summarized lesion ranging from 0 to 7 was determined for each APEC-challenged bird. Birds with lesions scoring 0-2 were regarded as mild infection, and those scoring 4-7 were designated as severe infection. The mild and severe pathology meant that birds were resistant and susceptible to APEC infection, respectively. Then, spleens from three groups, consisting of non-challenged, challenged-mild pathology and challenged-severe pathology were subjected to Solexa deep sequencing to investigate the dynamics of chicken miRNA expression.

first: Qinghua

last: Nie

first: Susan

last: Lamont

first: Xiquan

last: Zhang

first: Lisa

last: Nolan

first: Erin

last: Sandford


title: MicroRNA profiling of primary cutaneous large B-cell lymphomas

status: Public on Oct 18 2013

city: Leiden

first: Lianne

last: Koens

address: Albinusdreef 2

department: Pathology

country: Netherlands

institute: Leiden University Medical Center

summary: High-throughput sequencing of primary cutaneous follicle center lymphoma (PCFCL), primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) and in vitro activated peripheral blood B-cells. We performed high-throughput sequencing analysis on frozen tumor biopsies from 19 cases of PCFCL and PCLBCL-LT to establish microRNA profiles. Cluster analysis of the complete microRNome could not distinguish between the two subtypes, but 16 single microRNAs were found to be differentially expressed.

overall_design: Lymphoma miRNA profiles of were generated by deep sequencing, using Illumina Genome Analyzer II.

first: L

first: W

last: Leung

first: C

last: Tensen


title: Deep sequencing of endogenous mRNA from Caenorhabditis elegans in the presence of 2-deoxy-D-glucose (DOG) at 4 different time points

status: Public on Nov 06 2013

city: Jena

first: JenAge

last: Project

address: Beutenbergstr. 11

country: Germany

institute: Leibniz Institute for Age Research - Fritz Lipmann Institute

summary:
Background: C. elegans fed with a chemical inhibitor of glucose, namely 2-deoxy-D-glucose (DOG), exhibit considerably extended life span. DOG, in contradiction to D-glucose, cannot be metabolized in the glycolytic pathway. This results in the fact that less glucose is available for ATP production, and thus makes DOG-feeding of the roundworms equivalent to glucose restriction.
The RNA-seq data comprises 4 age groups (1, 5, 10 and 20 days  after L4)

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

overall_design: 11 samples: mRNA profiles of 1-day, 5-day and 10-day old worms as triplicates for DOG treatment; mRNA profiles of 20-day old worms as duplicates for DOG treatment

first: Steffen

last: Priebe

first: Uwe

last: Menzel

first: Kim

last: Zarse

first: Marco

last: Groth

first: Matthias

last: Platzer

first: Michael

last: Ristow

first: Reinhard

last: Guthke


title: Interplay Between Active Chromatin Marks and RNA-directed DNA Methylation in Arabidopsis thaliana

status: Public on Nov 01 2013

first: Christopher

middle: Joel

last: Hale

address: 1959 NE Pacific St., HSC H-458

institute: University of Washington

summary: DNA methylation is an epigenetic mark that is associated with transcriptional repression of transposable elements and protein coding genes. Conversely, transcriptionally active regulatory regions are strongly correlated with histone 3 lysine 4 di- and trimethylation (H3K4m2/3). We previously showed that Arabidopsis thaliana plants with mutations in the H3K4m2/m3 demethylase JUMONJI 14 (JMJ14) exhibit a mild reduction in RNA-directed DNA methylation (RdDM) that is associated with an increase in H3K4m2/m3 levels. To determine whether this incomplete RdDM reduction was the result of redundancy with other demethylases, we examined the genetic interaction of JMJ14 with another class of H3K4 demethylases: LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 1 and LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 2 (LDL1 and LDL2). Genome-wide DNA methylation analyses reveal that both families impact RdDM, but not other DNA methylation pathways. ChIP-seq experiments show that regions that exhibit an observable DNA methylation decrease are co-incidental with increases in H3K4m2/m3. Interestingly, the impact on DNA methylation was stronger at DNA-methylated regions adjacent to H3K4m2/m3-marked protein coding genes, suggesting that the activity of H3K4 demethylases may be particularly crucial to prevent spreading of active epigenetic marks. Finally, RNA sequencing analyses indicate that at RdDM targets, the increase of H3K4m2/m3 is not generally associated with transcriptional de-repression. This suggests that the histone mark itself—not transcription—impacts the extent of RdDM.

overall_design: For wild type plants (ecotype Columbia) and RdDM mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. The Col and nrpe1 BS-seq libraries were previously reported (GSE39247) and so are not part of this submission. In addition, two replicates of whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of nrpd1 mutant plants carrying a C-terminally epitope tagged (3XFLAG) NRPD1. Whole-genome bisulfite sequencing and small RNA sequencing was also performed on shh1 mutant plants transformed with the wild-type SHH1 protein-coding construct as well as multiple constructs containing point mutations. For these complementation libraries a separate shh1 mutant and Col control line were sequenced (“complementation replicates”).


title: Chromatin-to-nucleoprotamine transition is controlled by the histone H2B variant TH2B [ChIP-Seq]

city: La Tronche

first: Sophie

last: Rousseaux

address: Bd de la Chantourne

department: IAB

country: France

institute: INSERM-UGA

summary: The conversion of male germ cell chromatin to a nucleoprotamine structure is fundamental to the life cycle yet the underlying molecular details remain obscure. Here we show that an essential step is the genome-wide incorporation of TH2B, a histone H2B variant of hitherto unknown function. Using mouse models in which TH2B is depleted or C-terminally modified we show that TH2B directs the final transformation of dissociating nucleosomes into protamine-packed structures. Depletion of TH2B induces compensatory mechanisms that permit histone removal by up-regulating H2B and programming nucleosome instability through targeted histone modifications, including lysine crotonylation and arginine methylation. Furthermore, after fertilization, TH2B re-assembles onto the male genome during protamine-to-histone exchange. Thus, TH2B is a unique histone variant, which plays a key role in the histone-to-protamine packing of the male genome and guides genome-wide chromatin transitions that both precede and follow transmission of the male genome to the egg.

overall_design: Examination of TH2B binding on chromatin in meiotic (spermatocytes) and post-meiotic (round spermatids) male germ cells from adult Th2b+/tag mice (TH2B C-terminally fused to three consecutive affinity tags: His, Flag and Ha). Examination of TH2B (wild type) binding on chromatin in male germ cells from adult wt mice.

first: Saadi

last: Khochbin

first: Emilie

last: Montellier

first: Alexandra

last: Debernardi

first: Jean

last: Imbert

first: Hélène

last: Holota

first: Aurélie

last: Bergon

first: Fabrice

last: Lopez

first: Matthieu

last: Gérard

first: Fayçal

last: Boussouar


title: Genome Wide Profiling of p53 Response to Differentiation or  DNA Damage of Human Embryonic Stem Cells

city: Houston

first: Kadir

middle: Caner

last: Akdemir

state: TX

address: 1515 Holcombe Blvd

institute: MD Anderson Cancer Center

summary: Tumor suppressor p53 promotes differentiation of human embryonic stem cells (hESCs), but an in-depth understanding of mechanism is lacking. Here, we define p53 functions in hESCs by genome wide profiling of p53 chromatin interactions and intersection with gene expression during early differentiation and in response to DNA damage. During differentiation, p53 targets and regulates a unique collection of genes, many of which encode transcription factors and developmental regulators with chromatin structure poised by OCT4 and NANOG and marked by repressive H3K27me3 in pluripotent hESCs. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in regulation of cell death and cell cycle genes are conserved during both DNA damage and differentiation. Our findings expand the registry of p53 -regulated genes in hESCs and define specific functions of p53 in opposing pluripotency, which are highly distinct from stress-induced p53 response in stem cells.

overall_design: Identification of p53 binding sites in hESC under three conditions : Pluripotent, DNA damaged, Differentiating


title: Canonical Nucleosome Organization at Promoters Forms During Genome Activation

city: Shanghai

first: Yong

last: Zhang

address: 1239 Siping Road

institute: Tongji University

summary: The organization of nucleosomes influences transcriptional activity by controlling accessibility of DNA binding proteins to the genome. Genome-wide nucleosome binding profiles have identified a canonical nucleosome organization at gene promoters, where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. The mechanisms of formation and the function of canonical promoter nucleosome organization remain unclear. Here we analyze the genome-wide location of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear on thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization is independent of DNA sequence preference, transcriptional elongation, and robust RNA polymerase II (Pol II) binding. Instead, canonical promoter nucleosome organization correlates with the presence of Histone H3 Lysine 4 trimethylation (H3K4me3) and affects future transcriptional activation. These findings reveal that genome activation is central to the organization of nucleosome arrays during early embryogenesis.

overall_design: MNase-seq to generate nucleosome organization in two stages of zebrafish development; two biological replicates for each stage. 7 ChIP-seq experiments in three stages.  RNA-seq at 2.5 hpf (256-cell stage).

first: Nadine

middle: L

last: Vastenhouw

first: Jianxing

last: Feng

first: Kai

last: Fu

first: Chenfei

last: Wang

first: Ying

last: Ge

first: Andrea

last: Pauli

first: Paul

last: van Hummelen

first: Alexander

middle: F

last: Schier

first: Xiaole

last: Liu


title: Dynamic analysis of gene expression and genome wide transcription factor binding during lineage-specification of multipotent progenitors [ChIP-seq].

status: Public on Oct 17 2013

city: Lund

first: Shamit

last: Soneji

address: Sölvegatan 19

country: Sweden

institute: BMC

summary: We used the paradigmatic 'GATA-PU.1 axis’ to explore, at systems-level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIPSeq of GATA1, GATA2 and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (i)  differential complexity of sequence motifs bound by GATA1, GATA2 and PU.1; (ii) the scope and interplay of the GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (iii) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression and (iv) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry.  This 'rubric' exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.

overall_design: We examine binding of three factors Gata1, Gata2 and Pu1 at 3 stages of differentiation (multipotential, 5 days of erytroid and neutrophil) , while looking at 2 intermeditate stages of erythroid differentiation for factors Gata2 and Gata1. We also look at Gata2 and Gata1 binding after Gata1 induction using an ER fusion protein.

first: Tariq

last: Enver


title: M. tuberculosis (Mtb) Produces Nitrite in Human Macrophages in Physiologic Oxygen to Impact Mycobacterial ATP Consumption and Gene Expression

first: Genomics-core-facility

last: WCM

address: 1300 York Ave.

institute: Weill Cornell Medicine

summary: We sequenced mRNA from Mtb that had been treated with 5 mM nitrate or was untreated in standard 7H9/ADNaCl medium. This generated the first analysis of gene expression following mycobacterial nitrate respiration.

overall_design: Examination of mRNA levels in Mtb following nitrate respiration, which results in the production of nitrite.

first: Bussel

middle: A

last: Cunningham

first: T

first: C

last: Nathan


title: Comprehensive analysis of different in vitro insulin resistance models

city: Cambridge

first: Kinyui Alice

last: LO

state: MA

address: 77 Massachusetts Ave.

department: Biological Engineering

institute: MIT

summary: Diet-induced obesity (DIO) predisposes individuals to insulin resistance, and adipose tissue has a major role in the disease. Insulin resistance can be induced in cultured adipocytes by a variety of treatments, but what aspects of the in vivo responses are captured by these models remains unknown. We use global RNA sequencing to investigate changes induced by TNF-a, hypoxia, dexamethasone, high insulin, and a combination of TNF-a and hypoxia, comparing the results to the changes in white adipose tissue from DIO mice. We found that different in vitro models capture distinct features of DIO adipose insulin resistance, and a combined treatment of TNF-a and hypoxia is most able to mimic the in vivo changes. Using genome-wide DNase I hypersensitivity followed by sequencing, we further examined the transcriptional regulation of TNF-a-induced insulin resistance, and we found that C/EPBß key regulator of adipose insulin resistance.

overall_design: RNA-seq for 6 insulin resistance conditions and 2 control conditions, Dnase hypersensitivity-seq of 4 conditions and 1 control condition, ChIP-seq on p65 after TNFa treatment.

first: Kinyui


title: Global response to chemotherapy-induced apoptosis

first: Arun

middle: P.

last: Wiita

address: 185 Berry St., Suite 290

department: Laboratory Medicine

summary: Here we use an integrated systems-level examination of transcription, translation, and proteolysis to explore how cancer cells struggle with a chemotherapeutic drug prior to succumbing to apoptosis. As a model system we study myeloma cells exposed to the proteasome inhibitor bortezomib, a first-line clinical treatment. Despite robust transcriptional changes, unbiased quantitative proteomics detects production of only a few critical anti-apoptotic proteins against a background of general translation inhibition. Ribosome profiling further reveals potential translational regulation of stress response genes following bortezomib treatment. Once the apoptotic machinery is engaged, degradation by caspases is largely independent of changes at the transcriptional level. Moreover, previously uncharacterized non-caspase proteolytic events also participate in cellular deconstruction. As suggested by these data, we find that inhibition of the anti-apoptotic response regulator HSF1 promotes cell death by bortezomib. Thus, monitoring global cellular dynamics after chemotherapy offers in-depth insight into apoptosis and can also guide potential therapeutic combinations.

overall_design: We examined MM1.S myeloma cells exposed to 20 nM bortezomib across a time course with independent samples of poly(A) mRNA and ribosome footprints isolated at each of six time points (0h (untreated), 1.5h, 3h, 6h, 9h, 12h). Sequencing was performed on a Illumina HiSeq 2000 with single-end.

first: Etay

last: Ziv

middle: J

first: Anatoly

last: Urisman

first: Olivier

last: Julien

first: Alma

last: Burlingame

first: Jonathan

last: Weissman

first: James

last: Wells


title: Primate transcript and protein expression levels evolve under compensatory selection pressures

city: Tucson

first: Darren

middle: A.

last: Cusanovich

state: AZ

address: 1230 N Cherry Avenue, BSRL 421

department: Cellular and Molecular Medicine

institute: University of Arizona

summary: Variation in gene regulation is thought to have played an important role in the evolution of primates, and many studies have documented differences in mRNA expression levels across primate species. However, it is not yet known to what extent measurements of divergence in mRNA levels reflect divergence in protein expression levels, which are more directly tied to phenotypic differences. To address this question, we used high-resolution, quantitative mass spectrometry to collect thousands of protein expression measurements from human, chimpanzee, and rhesus macaque lymphoblastoid cell lines (LCLs). We also used RNA sequencing to collect transcript expression data from the same samples. Considering the two datasets jointly we found that there is much more inter-species divergence at the mRNA level than at the protein level. Remarkably, we found dozens of genes with significant expression differences between species at the mRNA level yet little or no difference in protein expression. Overall, our data suggest a much stronger evolutionary constraint on protein expression levels than on mRNA levels. We conclude that inter-species mRNA expression differences may often have limited functional consequences due to either buffering or compensatory changes in post-transcriptional or posttranslational regulation of proteins.

sample_organism:
Macaca mulatta
Pan troglodytes
Homo sapiens

overall_design: Gene expression patterns were compared between human, chimpanzee, and rhesus macaque lymphoblastoid cell lines (5 individuals from each species) using RNA-Seq. These gene expression patterns were also compared to protein expression patterns based on mass-spec data, which are available separately (see publication for details).

first: Zia

last: Khan

first: Michael

last: Ford

first: Amy

last: Mitrano

last: Pritchard

first: Yoav

last: Gilad


title: RNA-seq reveals changes in sphingolipid metabolism upon transformation

city: Copenhagen

first: Mikkel

last: Rohde

address: Strandboulevarden 49

country: Denmark

institute: Danish Cancer Society

summary: We sequenced mRNA from three independent pairs of mock and c-SRC transformed mouse embryonic fibroblasts and examined changes in expression of sphingolipid metabolism genes

overall_design: Three independent cultures of NIH3T3 MEFs were retrovirally transduced with either pBabe-Puro, og pBabe-Puro containing c-srcY527F. After a 7 day of selection in puromycine the three vector and three src transduced pools were propagated for RNA sequencing.

first: Marja

last: Jäättelä


title: Transcriptome analysis during somatic cell reprogramming

city: Kyoto

first: Takuya

last: Yamamoto

address: 53 Kawahara-cho, Shogoin, Sakyo-ku

department: CiRA

country: Japan

institute: Kyoto University

summary: By combining deep sequencing with high-throughput quantitative RT-PCR, we reveal that somatic splicing profiles are reorganized to pluripotent splicing profiles during reprogramming from mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. The splicing pattern in pluripotent stem cells resembles that in testis, and the regulatory regions have specific characters in length and sequence. These results indicate that the dynamic alteration in splicing is an integral part of the molecular network involved in the reprogramming process.

overall_design: Sequencing data from MEF, two iPS cell lines and one ES cell line. RT-PCR data will be represented within Figures or Tables in published manuscripts.

first: Sho

last: ohta


title: Genome-wide alteration of 5-hydroxymethylcytosine in a mouse model of fragile X-associated tremor/ataxia syndrome

city: Atlanta

first: Bing

last: Yao

state: GA

address: 615 Michael St. Rm 323

department: Human Genetics

institute: Emory University

summary: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder in which patients carry premutation alleles of 55-200 CGG repeats on the FMR1 gene. To date, whether alterations in epigenetic regulation modulate FXTAS has gone unexplored. 5-hydroxymethylcytosine (5hmC) converted from 5-methylcytosine (5mC) by the ten-eleven translocation (TET) family of proteins, has been found recently to play key roles in neuronal functions. Here we undertook genome-wide profiling of cerebellar 5hmC in a FXTAS mouse model (rCGG mice) and found that rCGG mice at 16 weeks showed overall reduced 5hmC levels genome-wide compared to age-matched wild-type littermates. However, we also observed gain-of-5hmC regions in repetitive elements, as well as cerebellum-specific enhancers, but not general enhancers. Genomic annotation and motif prediction of wild-type- and rCGG-specific differential 5-hydroxymethylated regions (DhMRs) revealed their high correlation with genes and transcription factors that are important in neuronal developmental and functional pathways. DhMR-associated genes partially overlapped with genes that were differentially associated with ribosomes in CGG mice identified by bacTRAP ribosomal profiling. Taken together, our data strongly indicate a functional role for 5hmC-mediated epigenetic modulation in the etiology of FXTAS, possibly through the regulation of transcription.

overall_design: Examination genome-wide 5hmC in FXTAS mice model

first: Yao

last: Bing

first: Jin

last: Peng


title: The response regulator ArcA uses a diverse binding site architechture to globally regulate carbon oxidation in E. coli [ChIP-Seq]

city: Madison

first: Dan

last: Park

state: WI

address: 440 Henry Mall

institute: UW Madison

summary: We found many binding sites for ArcA under glucose fermentative anaerobic growth conditions.  Descirbed in the manuscript "The response regulator ArcA uses a diverse binding site architechture to globally regulate carbon oxidation in E. coli"

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

overall_design: Examination of occupancy of ArcA under anaerobic growth conditions.

first: Sohail

last: Acktar

first: Aseem

last: Ansari

first: Robert

last: Landick

first: Patricia

last: Kiley


title: The response regulator ArcA uses a diverse binding site architechture to globally regulate carbon oxidation in E. coli

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

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

overall_design: Refer to individual Series


title: Genome-wide binding profiles of KLF3 and KLF3 mutants in MEF cells

city: Sydney

first: Jon

last: Burdach

state: NSW

address: UNSW

department: BABS

country: Australia

institute: University of New South Wales

summary: Transcription factors are often regarded as being comprised of a DNA-binding domain and a functional domain. The two domains are considered separable and autonomous, with the DNA-binding domain directing the factor to its target genes and the functional domain imparting transcriptional regulation. We have examined a typical Zinc Finger (ZF) transcription factor from the Krüppel-like factor (KLF) family, KLF3. This factor has an N-terminal repression domain that binds the co-repressor C-terminal binding protein (CtBP), and a DNA-binding domain composed of three classical (ZFs) at its C-terminus. We established a system to compare the genomic occupancy profile of wildtype KLF3 with two mutants affecting the N-terminal functional domain: a mutant unable to contact its cofactor CtBP and a mutant lacking the entire N-terminal domain, but retaining the ZFs intact. We used chromatin immunoprecipitation followed by sequencing (ChIP-seq) to assess binding across the genome in murine embryonic fibroblasts. Our results define the in vivo recognition site for KLF3 and the two mutants as a typical CACCC-like element. Unexpectedly, we observe that mutations in the N-terminal functional domain severely affect DNA binding. In general, both mutations reduce binding but there are also instances where binding is retained or even increased. These results provide a clear demonstration that the correct localization of transcription factors to their target genes is not solely dependent on their DNA-contact domains. This informs our understanding of how transcription factors operate and is of relevance to the design of artificial ZF proteins.

overall_design: ChIP-seq was performed on the three samples, KLF3, ΔDL and DBD in duplicate (biological replicates).  Input samples were used as controls.

first: Alister

last: Funnell

first: Crisbel

middle: M

last: Artuz

first: Mak

last: Sin

first: Lit

last: Tan

first: Richard

middle: C

last: Pearson

first: Merlin

last: Crossley


title: Sox2-regulatory networks in embryonic and trophoblast stem cells

city: Wako

first: Itoshi

last: NIKAIDO

state: Saitama

address: 2-1 Hirosawa

department: Center for Biosystems Dynamics Research

institute: RIKEN

summary:
Sox2 is a pleiotropic transcription factor that regulates self-renewal and differentiation capacity in different types of stem cells, raising the possibility that it regulates similar transcriptional programs controlling common stemness. Embryonic stem (ES) cells and trophoblast stem (TS) cells are two developmentally related types of stem cells, which originate from distinct lineages of peri-implantation embryos. We have found that Sox2 is a critical regulator of self-renewal in both of two stem cells. Genome-wide analysis of Sox2 target genes using Affymetrix Exon Arrays and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) unraveled that it regulates distinct transcriptional networks in ES and TS cells.
This SuperSeries is composed of the SubSeries listed below.


title: Genome-wide analyses of Fusarium fujikuroi reveal complex regulation of secondary metabolism and new metabolites

status: Public on Oct 16 2013

city: Corvallis

first: Kristina

last: Smith

state: OR

department: Biochemisty and Biophysics

institute: Oregon State University

summary: Fusarium fujikuroi is a biotechnologically important fungus due to its almost unique ability to produce gibberellic acids (GAs), a family of phytohormones. The fungus was described about 100 years ago as the causative agent of Bakanae (“foolish seedling”) disease of rice. Apart from GAs, the fungus is known to produce pigments and mycotoxins, but the biosynthetic genes are known for only eight products. Here we present a high-quality genome sequence of the first member of the Gibberella fujikuroi species complex (GFC) that allowed de novo genome assembly with 12 scaffolds corresponding to the 12 chromosomes. In this work we focused on identification of all potential secondary metabolism-related gene clusters and their regulation in response to nitrogen availability by transcriptome, proteome, HPLC-FTMS and ChIP-seq analyses. We show that most of the cluster genes are regulated in a nitrogen-dependent manner, and that expression profiles fit to proteome and ChIP-seq data for some but not all clusters. Comparison with genomes of all available Fusarium species, including the recently sequenced F. mangiferae and F. circinatum, showed only a small number of common gene clusters and provides new insights into the divergence of secondary metabolism in the genus Fusarium. Phylogenetic analyses suggest that some gene clusters were acquired by horizontal gene transfer, while others were present in ancient Fusarim species and have evolved differently by gene duplications and losses. One polyketide synthase (PKS) and one non-ribosomal peptide synthetase (NRPS) gene cluster are unique for F. fujikuroi. Their products were identified by combining overexpression of cluster genes with HPLC-FTMS-based analyses. In planta expression studies suggest a specific role of the PKS19 product in rice infection. Our results indicate that comparative genomics together with the used genome-wide experimental approaches is a powerful tool to uncover new secondary metabolites and to understand their regulation at the transcriptional, translational and epigenetic levels.

overall_design: Examination of 3 different histone modifications, with 2 growth conditions for one of the modifications (Total of 4 samples)

first: P

last: Wiemann

first: C

last: Sieber

first: K

last: von Bargen

first: L

last: Studt

first: E

last: Niehaus

last: Huss

last: Michielse

first: S

last: Albermann

first: D

last: Wagner

first: J

last: Espino

middle: V

last: Bergner

middle: R

last: Connolly

first: A

last: Fischer

first: G

last: Reuter

last: Kleigrewe

first: T

last: Bald

first: B

last: Wingfield

first: R

last: Ophir

last: Freeman

first: M

last: Hippler

last: Brown

middle: H

last: Proctor

last: Münsterkötter

last: Freitag

first: H

middle: U

last: Humpf

first: U

last: Güldener

last: Tudzynski


title: Dynamics of Sox2 and Esrrb occupancy during the differentiation of embryonic stem cells into trophoblast stem cells.

first: Itoshi

last: NIKAIDO

summary: To understand the mechanism underlying the versatility in transcriptional regulation by Sox2 and Esrrb, we compared genome-wide binding sites of Sox2 and Esrrb in embryonic stem (ES) cells and trophoblast stem (TS) cells by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).

overall_design: A tetracycline-inducible Oct3/4 knockout ES cell line ZHBTc4 was treated with Tet for 4 days in the presence of FGF4 and mouse embryonic fibroblasts (MEFs).

first: Kenjiro

last: Adachi

last: Nikaido

first: Hiroki

last: Ura

last: Ueda

first: Hitoshi

last: Niwa


title: Synthetic cationic peptide IDR-1018 modulates human macrophage differentiation

city: Vancouver

first: Christopher

last: Fjell

state: BC

address: Room 166 - 1081 Burrard Street

department: Medicine

institute: University of British Columbia

summary: Synthetic, innate defense regulators (IDR) peptides, designed based on natural host defenses peptides, have enhanced immunomodulatory activities and reduced toxicity leading to protection in infection and inflammation models that is dependent on macrophages/monocytes. Here we measured the effect of IDR-1018 on macrophage gene expression during differentiation. Differentiation in the presence of IDR-1018 induced a unique signature of immune responses suggesting that IDR-1018 drives macrophage differentiation towards an intermediate M1-M2 state, enhancing anti-inflammatory functions while maintaining certain pro-inflammatory activities important to the resolution of infection.

overall_design: RNA-seq was performed using the Illumina Genome Analyzer IIx platform. Monocytes were isolated from 3 healthy donors, and left unstimulated  or stimulated for 4 hours with 20 μg/ml IDR-1018. For library preparation, 500 ng of total RNA was processed according to the Illumina TruSeq RNA sample preparation guide (Illumina catalogue number FC-122-1002). Briefly, mRNA was purified using poly-dT beads, followed by synthesis of the first and second cDNA strands, end repair addition of an poly-A overhang, and ligation of adapters and unique barcodes, as per the manufacturer’s instructions. DNA enrichment was carried out via a 15-cycle PCR. Following quantification, 8 pM of dsDNA was used for cluster generation on a CBOT instrument (Illumina, San Diego, CA). RNA sequencing was done on a GAIIx instrument (Illumina), performed as a single read run with 51 amplification cycles. Data processing was carried out in house, using CASAVA to convert raw data and demultiplex to FASTQ sequence files. Reads were aligned to the reference genome using TOPHAT, and then mapped to genes using the Bioconductor package GenomeRanges.

first: Olga

last: Pena

first: Nicole

last: Afacan

first: Jelena

last: Pistolic

first: Carol

last: Chen

first: Reza

last: Falsafi

middle: D

first: Robert

middle: E

last: Hancock


title: Quantitative assessment of single-cell RNA sequencing methods

city: Stanford

first: Angela

middle: Ruohao

last: Wu

state: CA

address: 318 Campus Drive, Clark Center E300

department: Bioengineering

institute: Stanford University

summary: We generated single-cell transcriptomes from a large number of single cells using several commercially available platforms, in both microliter and nanoliter volumes, and compared performance between them. We benchmarked each method to conventional RNA-seq of the same sample using bulk total RNA, as well as to multiplexed qPCR, which is the current gold standard for quantitative single-cell gene expression analysis. In doing so, we were able to systematically evaluate the sensitivity, precision, and accuracy of various approaches to single-cell RNA-seq.  Our results show that it is possible to use single-cell RNA-seq to perform quantitative transcriptome measurements of individual cells, that it is possible to obtain quantitative and accurate gene expression measurements with a relatively small number of sequencing reads, and that when such measurements are performed on large numbers of cells, one can recapitulate the bulk transcriptome complexity, and the distributions of gene expression levels found by single-cell qPCR.

overall_design: 109 single-cell human transcriptomes were analyzed in total; 96 using nanoliter volume sample processing on a microfluidic platform, Nextera library prep (biological replicates); 3 using the SMARTer cDNA synthesis kit, Nextera library prep (biological replicates); 3 using the Transplex cDNA synthesis kit, Nextera library prep (biological replicates); 7 using the Ovation Nugen cDNA synthesis kit (biological replicates) where 3 used Nextera library prep and 4 used NEBNext library prep. In addition, 4 bulk RNA samples were sequenced: bulk RNA generated using ~1 million pooled cells was used to make bulk libraries, 2 of which were made using SMARTer cDNA synthesis kit (technical replicates) and 2 made using Superscript RT kit with no amplification (technical replicates). All 4 bulk samples were made into libraries using Nextera.

first: Stephen

last: Quake


title: Chromatin-to-nucleoprotamine transition is controlled by the histone H2B variant TH2B

status: Public on Oct 15 2013

first: Sophie

last: Rousseaux


title: Rrp5 binding at multiple sites coordinates pre-rRNA processing and assembly

status: Public on Oct 14 2013

city: PARIS

first: Simon

last: Lebaron

address: 4 avenue de l'observatoire

institute: Université Paris Descartes

summary: The CRAC UV crosslinking technique identified numerous pre-rRNA binding sites for the large, highly conserved ribosome synthesis factor Rrp5. Intramolecular complementation has shown that the C-terminal domain (CTD) of Rrp5 is required for pre-rRNA cleavage at sites A0-A2 on the pathway of 18S rRNA synthesis, whereas the N-terminal domain (NTD) is required for A3 cleavage on the pathway of 5.8S/25S rRNA synthesis. The CTD was crosslinked to sequences flanking A2 and to the snoRNAs U3, U14, snR30 and snR10, which are required for cleavage at A0-A2. The NTD was crosslinked to the sequence flanking A3 and to the RNA component of RNase MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural protein factors and NTPases. A key role in coordinating pre-ribosomal assembly and processing was confirmed by "Miller" chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and pre-ribosome compaction greatly reduced.

overall_design: CRAC results are provided for four individual samples: Rrp5 Full-length (FL) in vivo, Rrp5 Full-length (FL) in vitro, Rrp5 NTD and Rrp5 CTD.

first: Asa

last: Segerstolpe

first: Sander

last: Grenneman

first: Lars

last: Wieslander

first: David

last: Tollervey


title: Subcellular RNA Sequencing Reveals Broad Presence of Cytoplasmic Intron-sequence Retaining Transcripts in Mouse and Rat Neurons.

city: Philadelphia

first: Junhyong

last: Kim

state: PA

address: 433 S University Avenue

department: Biology

institute: University of Pennsylvania

summary:
Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells.
One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons.
We hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation.
We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions.
Two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons were sequenced.
After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species  (44-60% of the expressed transcripts).
The analysis for CIRTs was also carried out by sequencing single cells from mouse brown adipose tissue and mouse cardiomyocytes. There was widespread prevalence of CIRTs in all of the cell types.

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: Single cell samples were aRNA amplified and sequenced using Illumina GA Analyzer II and Illumina Hiseq 2000

first: Jim

last: Eberwine

first: Bernhard

last: Kuhn

first: Mugdha

last: Khaladkar


title: Small RNA from Arabidopsis thaliana with RNA silencing directed to Chalcone Synthase (At5G13930) by a hairpin RNA, a 21-nt artificial miRNA and a 22-nt artificial miRNA in the pap1-D over-expressing background

first: Marcus

last: McHale

address: Lvl 8 Biochemistry Bldg G08, University of Sydney

department: School of Molecular Biology

institute: University of Sydney

summary: 22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger trans-acting phased small interfering RNA (tphasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA) mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA DEPENDENT RNA POLYMERASE6 mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families.

overall_design: sRNA sequencing from aerial tissues of 3-week old plants grown on MS media

first: Andrew

last: Eamens

first: E

last: Finnegan

first: Peter

last: Waterhouse


title: Extended self-renewal and accelerated reprogramming in the absence of Kdm5b [RNA-Seq]

city: Detroit

first: Benjamin

last: Kidder

state: MI

address: 4100 John R St

department: Oncology

institute: Wayne State University

summary: ES cell pluripotency is thought to be regulated in part by H3K4 methylation. However, it is unclear how H3K4 demethylation contributes to ES cell function and participates in iPS cell reprogramming. Here, we show that KDM5B, which demethylates H3K4, is important for ES cell differentiation, and presents a barrier to the reprogramming process. Depletion of Kdm5b leads to an extension in the self-renewal of ES cells in the absence of LIF. Transcriptome analysis revealed the persistent expression of pluripotency-genes and underexpression of developmental genes during differentiation in the absence of Kdm5b, suggesting that KDM5B plays a key role in cellular fate changes. We also observed accelerated reprogramming of differentiated cells in the absence of Kdm5b, demonstrating that KDM5B is a barrier to the reprogramming process. Expression analysis revealed that mesenchymal master regulators associated with epithelial-to-mesenchymal transition (EMT) are downregulated during reprogramming in the absence of Kdm5b. Moreover, global analysis of H3K4me3/2 revealed that enhancers of fibroblast genes are rapidly deactivated in the absence of Kdm5b, and genes associated with EMT lose H3K4me3/2 during the early reprogramming process. These findings provide functional insight into the role for KDM5B in regulating ES cell differentiation and as a barrier to the reprogramming process.

overall_design: RNA-Seq of undifferentiated and embryoid body (EB) differentiated murine shLuc and shKdm5b ES cells

first: Gangqing

last: Hu

first: Keji

last: Zhao


title: Ribosome profiling reveals features of normal and disease-associated mitochondrial translation

city: Amsterdam

first: Koos

last: Rooijers

address: Plesmanlaan 121

department: Gene Regulation

institute: Netherlands Cancer Institute

summary: Ribosome profiling and RNAseq data on human BJ fibroblasts and cybrid cells using an adapted ribosome profiling protocol to improve detection of mitochondrial ribosome protected fragments

overall_design: 51-base length single read ribosome profiling data on human fibroblasts and cybrid cells using an adapted ribosome profiling protocol; and 51-base length single read RNAseq on polyA enriched RNA

first: Fabricio

last: Loayza-Puch

first: Leo

middle: G

last: Nijtmans

first: Reuven

last: Agami


title: Extended self-renewal and accelerated reprogramming in the absence of Kdm5b [ChIP-Seq]

overall_design: ChIP-Seq for H3K4me3 and H3K4me2 in murine shLuc and shKdm5b 4TF-MEFs (tetO-Pou5f1,-Sox2,-Klf4,-c-Myc) at 48h.


title: Dynamic analysis of gene expression and genome wide transcription factor binding during lineage-specification of multipotent progenitors

first: Shamit

last: Soneji

address: Sölvegatan 19

institute: BMC

first: Tariq

last: Enver


title: Genome-wide localization of Smc1, Smc3 and CTCF in mouse B cells.

status: Public on Oct 11 2013

city: Illkirch

first: Anne-Sophie

last: THOMAS

address: 1 rue Laurent Fries

institute: IGBMC

summary: Immunoglobulin class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation induced cytidine deaminase (AID) to switch regions and by the subsequent generation and resolution of dsDNA breaks (DSBs). During, CSR the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers and switch regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. Here we show that during CSR, AID associates with subunits of cohesin, a complex previously implicated in DNA repair and in the formation of DNA loops between enhancers and promoters. By ChIP-Seq experiments, we find that Cohesin is dynamically recruited to the IgH locus during CSR and that knockdown of Cohesin or its regulatory subunits results in impaired CSR and abnormal DSB resolution. Our results are consistent with a model in which Cohesin controls the formation of long-range DNA loops at the IgH locus and the resolution of DSBs generated during CSR.

overall_design: Smc1, Smc3 and CTCF were immunoprecipitated from resting or in vitro activated B cells.

last: Thomas-Claudepierre

first: Ebe

last: Schiavo

first: Vincent

last: Heyer

first: Marjorie

last: Fournier

first: Isabelle

last: Robert

first: Bernardo

last: Reina San-Martin


title: Differences in CTCF binding site sequence are associated with unique regulatory and functional trends during embryonic stem cell differentiation [RNA-Seq]

city: Boston

first: Sebastien

last: Vigneau

address: 450 Brookline Avenue

department: Cancer Biology

institute: Dana-Farber Cancer Institute

summary: CTCF (CCCTC-binding factor) is a highly conserved 11-zinc finger DNA binding protein with tens of thousands of binding sites genome-wide. CTCF acts as a multifunctional regulator of transcription, having been previously associated with activator, repressor, and insulator activity. These diverse regulatory functions are crucial for preimplantation development and are implicated in the regulation of numerous lineage-specific genes.  Despite playing a critical role in  developmental gene regulation, the mechanisms that underlie developmental changes in CTCF recruitment and function are poorly understood. Our previous work suggested that differences in CTCF’s binding site sequence may affect the regulation of CTCF recruitment, as well as CTCF’s regulatory function. To investigate these two possibilities directly during a developmental process, changes in genome-wide CTCF binding and gene expression were characterized during in vitro differentiation of mouse embryonic stem cells. CTCF binding sites were initially separated into three classes (named LowOc, MedOc, and HighOc) based on similarity to the consensus motif.  The LowOc class, with lower-similarity to the consensus motif, is more likely to show changes in binding during differentiation.  These more dynamically bound sites are enriched for motifs that confer a lower in vitro affinity for CTCF, suggesting a mechanism where sites with low-binding affinity are more amenable to developmental control. Additionally, by comparing changes in CTCF binding with changes in gene expression during differentiation, we show that LowOc and HighOc sites are associated with distinct regulatory functions.  In sum, these results suggest that the regulatory control of CTCF’s binding and function is dependent in part upon specific motifs within its DNA binding site.

overall_design: Mouse E14 ES cells were differentiated in vitro for 4.5 days using retinoic acid. RNA-Seq was performed from cells collected before and after differentiation.

first: Robert

middle: N

last: Plasschaert

first: Sébastien

first: Italo

last: Tempera

first: Ravi

last: Gupta

first: Jasna

last: Maksimoska

first: Logan

last: Everett

first: Ramana

last: Davuluri

first: Ronen

last: Mamorstein

first: Paul

last: Lieberman

first: David

last: Schultz

first: Sridhar

last: Hannenhalli

first: Marisa

middle: S

last: Bartolomei


title: Differences in CTCF binding site sequence are associated with unique regulatory and functional trends during embryonic stem cell differentiation


title: RNA-seq of Arabidopsis spl7 identifies two genes required for copper acquisition and a copper dependence of iron homeostasis

city: Bochum

first: Ute

last: Kramer

address: Gebaude ND Nord Raum 3/30, Universitatsstrase 150

department: Department of Plant Physiology

institute: Ruhr University Bochum

summary: The transition metal copper (Cu) is an essential element for all living organisms. In plants, Cu plays key roles in electron transport chains of chloroplasts and mitochondria, as well as in cell wall metabolism, ethylene perception, molybdenum cofactor synthesis and oxidative stress protection. Because of its physiological importance, suboptimal concentrations of Cu trigger a re-organization of metabolism to economize on Cu, and pronounced Cu deficiency causes severe growth reduction and defects in male fertility. However, when present in excess, Cu can be highly toxic. Therefore, Cu uptake, utilization and cellular concentrations are strictly regulated. A number of components of the Cu-homeostatic network of Arabidopsis have already been identified. However, the mechanisms that control responses of plant gene expression to Cu-deficiency are only partly understood. In Chlamydomonas reinhardtii, the transcription factor Crr1 is required for activating and/or repressing the expression of a number of genes in response to Cu deficiency. This protein contains a plant-specific DNA-binding domain (SBP domain), ankyrin repeats and a C-terminal cysteine-rich region with similarity to a Drosophila metallothionein (MT). In Arabidopsis, there is a family of 16 proteins with SBP domains named SPL family (SBP-like) of which a subset of proteins have been implicated in flowering time control and floral development. Among all of them, AtSPL7 is the most similar to Crr1 (27 % identity), and AtSPL1 (25 % identity), AtSPL12 (24 % identity) and AtSPL14 (23 % identity) share a common protein architecture. The goal of this work was to obtain a complete account of the response of the Arabidopsis thaliana transcriptome to Cu deficiency, as well as  to determine the role of AtSPL7 in the transcriptional response to Cu deficiency. For this purpose, three independent experiments were carried out including Col-0 wild-type and spl7-2 mutant plants grown in Cu-sufficient and Cu-deficient hydroponic media, respectively. Root and shoot transcriptomes were established by RNA-Seq for quantitative comparisons.

overall_design: Sampling of root and shoot tissues of wild-type (WT, Col-0) and spl7-2 mutant plants (the mutant is knock-down for the transcription factor SPL7, which plays a key role in the transcriptional regulation of the copper deficiency responses) cultivated hydroponically in a modified Hoagland's solution under Cu-sufficient (control) and Cu-deficient conditions.

first: Maria

last: Bernal

first: Vasantika

last: Singh

last: Casero

first: Arne

last: Grande

first: Peter

last: Huijser

first: Matteo

last: Pellegrini

first: Sabeeha

last: Merchant

last: Kraemer


title: Extended self-renewal and accelerated reprogramming in the absence of Kdm5b

first: Benjamin

middle: L

last: Kidder

first: Gangqing

last: Hu

first: Keji

last: Zhao


title: Epigenetic modifications induced by Blimp-1 regulate CD8+ T cell memory progression during acute virus infection

status: Public on Oct 10 2013

city: Seoul

first: Hyun Mu

last: Shin

address: Biomedical Science Building 202, 103 Daehakro, Jongro-gu

department: Biomedical Sciences

country: South Korea

institute: Seoul National University College of Medicine

summary: Blimp-1 regulates the overall accumulation of virus-specific CD8+ T cells during acute viral infections.  Increased proliferation and survival of Blimp-1-deficient CD8+ T cells is promoted by persistent cytokine responsiveness, resulting from sustained expression of CD25 and CD27.  Knockdown of these genes reduced the Blimp-1-deficient CD8+ T cell response.  Genome-wide ChIP-sequencing analysis identified CD25 and CD27 genes as direct targets of Blimp-1.  At the peak of the anti-viral response, but not earlier, Blimp-1 recruited the histone modifying enzymes G9a and HDAC2 to the Il2ra and Cd27 loci, thereby repressing expression of these genes.  In the absence of Blimp-1, the Il2ra and Cd27 genes exhibited enhanced histone H3-acetylation and reduced histone H3K9-trimethylation.  These data elucidate a central mechanism by which Blimp-1 acts as an epigenetic regulator, enhancing the numbers of short-lived effector cells while suppressing the development of memory precursor CD8+ T cells.

overall_design: Naïve CD8+ T cells from OT-I TCR transgenic Rag1-/- mice were stimulated with anti-CD3 and anti-CD28 for three days in vitro, in the presence of IL-2 to up-regulate Blimp-1 protein. Genome-wide mapping of Blimp-1 binding in mouse CD8+ T cells was conducted.

first: H

first: V

last: Kapoor

first: T

last: Guan

first: S

last: Kaech

first: R

last: Welsh

first: L

last: Berg


title: Gene regulation and priming by Topoisomerase IIα in embryonic stem cells [FAIRE-Seq]

city: Cambridge

first: Sudhir

last: Thakurela

address: 7 Divinity Avenue

institute: HSCRB

summary: Topoisomerases are essential for resolving topological problems in the genome, while their function in gene regulation, especially during cellular differentiation, remains unknown. We reveal that the expression of two Topo II isoforms, Top2a and Top2ß, is characteristic of dividing and postmitotic tissues, respectively. In embryonic stem cells, Top2a preferentially binds to promoters embedded in an active chromatin environment. Inhibition of Top2a activity results in misregulation of target gene expression that accompanies accumulation of double-strand breaks. Common targets of Top2a and Top2ß are housekeeping genes while their unique targets are involved in proliferation/pluripotency and neurogenesis, respectively. Moreover, a subset of Top2a targets exhibit bivalent chromatin state that is resolved upon differentiation concomitant with their activation and occupancy by Top2ß, a feature further observed for long genes. These findings suggest that Top2a not only contributes to stem cell transcriptome regulation but may also prime developmental genes for subsequent activation upon differentiation.

overall_design: mRNA profiles of DMSO and ICRF-193 treated mESCs were generated by deep sequencing in triplicates. ICRF-193 is a well established catalytic inhibitor of Topoisomerase II, hence, we used ICRF-193 to the elucidate role of Top2a catalytic activity on transcription by genome wide transcription profiling.

first: Angela

last: Garding

first: Johannes

last: Jung

first: Lukas

last: Burger

first: Vijay

last: Tiwari


title: Gene regulation and priming by Topoisomerase IIα in embryonic stem cells

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

overall_design: Refer to individual Series


title: Gene regulation and priming by Topoisomerase IIα in embryonic stem cells [RNA-Seq]


title: RNA-seq transcriptome analysis of Nestin-GFP-peri and -GFP-retic bone marrow stromal cells

city: Bronx

first: Paul

last: Frenette

state: NY

address: 1301 Morris Park Avenue , Room 101

department: Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research

institute: Albert Einstein College of Medicine

summary: Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSC in the bone marrow (BM) remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging technique and computational modelling to analyse significant tridimensional associations among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal BM. These arterioles are ensheathed exclusively by rare Nestin-GFP-peri/NG2+ pericytes, distinct from sinusoid-associated Nestin-GFP-retic/LepR+ cells. The present RNA-seq study sought to obtain a comprehensive understanding of the differences between the two distinct HSC cellular niches.

overall_design: mRNA profiles of sorted Nestin-GFP-peri and -GFP-retic bone marrow stromal cells were generated from pooled mice in triplicate by Illumina HiSeq 2000 sequencing.

first: Qiaozhi

last: Wei


title: Identification of novel potato candidate genes for quantitative resistance to Phytophthora infestans by SuperSAGE transcriptome analysis

city: Köln

first: Christiane

last: Gebhardt

address: Carl-von-Linné-Weg 10

institute: Max Planck Institute for Plant Breeding Research

summary: To gain novel molecular insights into quantitative late blight resistance, we performed a high-resolution quantitative analysis of gene expression using potato cultivars with contrasting SNP alleles at the StAOS2 locus associated with maturity corrected resistance (MCR). SuperSAGE samples were generated from uninfected and infected plants of the selected genotypes under controlled environmental conditions. Genotypes were pooled to reduce the influence of the genetic background on the transcriptome. Nine SuperSAGE samples were prepared from artificially inoculated plants in a growth chamber using total RNA of the pooled 14, 6 and 9 genotypes in groups A1, A2 and B2, respectively, at three infection time points T0, T1 and T2. Combining the tag counts of both NlaIII and DpnII libraries resulted in 1.1 to 6.2 million tags per sample. Of total 266361 unique tags (unitags), 52.6% matched to the potato genome sequence when up to three mismatches per 26 base pairs were allowed, and 23.3% matched without mismatch. Fifteen pair wise comparisons were performed between the nine SuperSAGE samples to identify transcripts that were differentially expressed in response to infection (six comparisons) or between three genotype pools at the infection time points T0, T1 and T2 (nine comparisons). The number of unitags per comparison ranged from 127 000 to 182 000 (average 158 000). Between 2100 and 11800 tags were differentially expressed in pair wise comparisons, depending on arbitrary cut-off p-values for a significant difference. The highest number of differences was observed for the comparison between genotype pools A1 and A2 one day after infection (A1-T1 vs A2-T1), and the lowest between genotype pools A2 and B2 two days after infection (B2-T2 vs A2-T2). The number of differences in response to infection and between genotype pools even before infection (T0) was in the same order of magnitude. Based on the annotations in the DFCI potato gene index, in the potato genome and in few cases by BLASTX searches against the protein database at NCBI, transcripts that showed reproducible differential expression over the infection time course or between genotype pools A1, A2 and B2 were grouped in 16 functional categories, with overlaps between categories. Genes with genotype dependent, constitutive differential expression provide excellent targets for developing novel diagnostic markers for breeding cultivars with improved quantitative resistance to late blight and possibly other biotic and abiotic stresses. Relevant in this respect appear, besides numerous genes of unknown or ill-defined function, genes with known function involved in stress responses, photosynthesis, protein biosynthesis, protein degradation via the 26S proteasome, transport of proteins, lipids, ions and other small molecules, cell wall structure and many others.

overall_design: Eighteen SuperSAGE libraries were constructed based on nine leaf samples from one infection experiment. For each time point (T0, T1 and T2) one leaflet each of 14, 6 and 9 SL genotypes in genotypic groups A1, A2 and B2, respectively, were pooled. Frozen pooled leaf tissue was powdered in a CryoMill. SuperSAGE libraries were generated at GenXPro GmbH (Frankfurt, Germany) essentially as described (Matsumura et al. 2010). To prevent amplification biases the TrueQuant technology was applied as described by B. Rotter (Patent application Nr. WO2009152928). Besides NlaIII (recognition site: 5’-CATG-3’), DpnII (recognition site: 5’-GATC-3’) was used as second anchoring enzyme, to capture transcripts without a NlaIII site. Therefore, the 26 bp tags carry either CATG or GATC at their 5’ end. The libraries were pooled and sequenced by Solexa/Illumina technology (Illumina, Inc., USA).

first: Astrid

last: Draffehn

first: Li

last: Li

first: Nicolas

last: Krezdorn

first: Jia

last: Ding

first: Jens

last: Lübeck

first: Josef

last: Strahwald

first: Meki

last: Muktar

first: Birgit

last: Walkemeier

first: Björn

last: Rotter


title: Antiviral RNA interference in mammalian cells

city: Zurich

first: Antonin

last: Marchais

address: Universitatstrasse 2

department: Department of Biology

institute: ETHZ

summary: Plants and invertebrates protect themselves from viruses through RNA interference (RNAi), yet it remains unknown whether this defense mechanism exists in mammals. Antiviral RNAi involves the processing of viral long double-stranded (ds) RNA molecules into small interfering RNAs (siRNAs) by the ribonuclease (RNAse) III Dicer. These siRNAs are incorporated into effector complex(es) containing members of the Argonaute (Ago) protein family and guide silencing of complementary target viral RNAs. Here, we detect the accumulation of phased Dicer-dependent virus-derived siRNA (viRNAs) and demonstrate their loading into Ago2 after infection of mouse embryonic stem (ES) cells with Encephalomyocarditis virus (EMCV). We further show that the production of these viRNAs is drastically reduced, yet not completely abolished, if ES cells are first induced to differentiate before infection. Finally, we reveal that the mammalian virus Nodamura virus (NoV) encodes for a protein that counteracts such antiviral RNAi in ES cells supporting the existence of an effective RNAi-based immunity in mammals.

overall_design: Infection of wild-type or mutant mouse ES cells and analysis of small RNAs from total extracts or immunoprecipitated components of the RNAi pathway

first: Pierre

last: Maillard

first: Constance

last: Ciaudo

first: Yang

first: Florence

last: Jay

first: Shou-Wei

first: Olivier

last: Voinnet


title: Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer [RRBS]

city: Seattle

first: Scott

middle: J.

last: Diede

state: WA

address: 1100 Fairview Avenue North, Mailstop c3-168

institute: Fred Hutchinson Cancer Research Center

summary: Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared to the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Examination of DNA methylation in one representative human medulloblastoma patient sample and three different mouse models of medulloblastoma using RRBS

first: Zizhen

last: Yao

first: C

last: Keyes

first: Ashlee

last: Tyler

first: Joyoti

last: Dey

first: Christopher

last: Hackett

first: Katrina

last: Elsaesser

last: Kemp

first: Paul

last: Neiman

first: William

middle: A

last: Weiss

first: James

last: Olson

first: Stephen

last: Tapscott


title: Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast [RNA-seq]

status: Public on Oct 09 2013

first: Peter

middle: J

last: Park

address: 10 Shattuck St

department: Center for Biomedical Informatics

institute: Harvard Medical School

summary: Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression.  To gain a comprehensive understanding of the requirements for this critical factor, we have performed genome-wide analyses of transcription, chromatin structure, and histone modifications in an S. pombe spt6 mutant.  Our results demonstrate several dramatic changes to transcription and chromatin structure in the spt6 mutant, including an elevation of antisense transcripts at over 70 percent of all genes and general loss of the +1 nucleosome.  Furthermore, Spt6 is required for the trimethylation of histone H3 on lysines 4 and 36, marks associated with active transcription.  Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones.

overall_design: RNA-seq experiments were performed on wild type and spt6-1 strains in replicate

first: Christine

last: DeGennaro

first: Burak

last: Alver

first: Fred

last: Winston


title: NCoR Repression of LXRs Restricts Macrophage Biosynthesis of Insulin-Sensitizing Omega 3 Fatty Acids

city: La Jolla

first: Nathanael

middle: Judge

last: Spann

address: 9500 Gilman Drive CMMW 219A

department: Cellular and Molecular Medicine

institute: UCSD

summary: Macrophage-mediated inflammation is a major contributor to obesity-associated insulin resistance. The corepressor NCoR interacts with inflammatory pathway genes in macrophages, suggesting that its removal would result in increased activity of inflammatory responses. Surprisingly, we find that macrophage-specific deletion of NCoR instead results in an anti-inflammatory phenotype along with robust systemic insulin sensitization in obese mice. We present evidence that de-repression of LXRs contributes to this paradoxical anti-inflammatory phenotype by causing increased expression of genes that direct biosynthesis of palmitoleic acid and ω3 fatty acids. Remarkably, the increased ω3 fatty acid levels primarily inhibit NF-κB-dependent inflammatory responses by uncoupling NF-κB binding and enhancer/promoter histone acetylation from subsequent steps required for pro-inflammatory gene activation. This provides a mechanism for the in vivo anti-inflammatory insulin sensitive phenotype observed in mice with macrophage-specific deletion of NCoR. Therapeutic methods to harness this mechanism could lead to a new approach to insulin sensitizing therapies.

overall_design: ChIP-Seq and Gro-Seq profiling was performed in thioglycollate-elicited peritoneal macrophages treated as indicated.

first: Pingping

last: Li

first: Minna

last: Kaikkonen

first: Min

last: Lu

first: Da-Young

last: Oh

first: Jesse

last: Fox

first: Gautam

last: Bandyopadhyay

first: Saswata

last: Talukdar

first: Jianfeng

last: Xu

last: Lagakos

first: David

last: Patsouris

first: Aaron

last: Armando

first: Oswald

last: Quehenberger

first: Edward

last: Dennis

first: Steven

last: Watkins

first: Johan

last: Auwerx

middle: K

last: Glass

first: Jerrold

last: Olefsky


title: Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast [ChIP-seq]

first: Peter

last: Park

overall_design: ChIP-seq experiments were performed on wild type and spt6-1 strains on the following proteins: RNA polymerase II (Rpb1), Paf1 Complex (Ctr9), COMPASS (Swd1), Set2, Spt6, histones H2B and H3, histone modifications H3K4me3 and H3K36me3. Experiments were performed in replicates and matching inputs were also sequenced.

first: Christine

middle: M

last: DeGennaro

first: Burak

last: Alver

first: Fred

last: Winston


title: Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast


title: Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

first: Scott

last: Diede

type: Methylation profiling by genome tiling array


title: Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast [Mnase-seq]

overall_design: MNase-seq experiments were performed on wild type and spt6-1 strains in replicate at two different MNase concentrations


title: Spliceosome-Mediated-Decay (SMD) regulates expression of non-intronic genes in budding yeast

city: Edinburgh

first: Sander

last: Granneman

address: Mayfield Road, Kings Buildings, Waddington building, room 3.06

department: Centre for Synthetic and Systems Biology

country: United Kingdom

institute: University of Edinburgh

summary:
Purpose: The goals of this study were to determine whether the spliceosome interacts with non-intronic mRNAs

Methods: RNAseq was performed on RNA that immunoprecipitated with the yeast SMD1 protein. Tandem-affinity-purified RNAs were extracted and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). We also performed RNAseq experiments on rRNA depleted total RNA extracted from an exosome mutant (rrp6Δ), a temperature-sensitive splicing mutant (prp40-1) and a parental strain (BY4741). The rRNA was depleted using the Invitrogen RiboMinus kit, according to manufactureres procedures. The depleted RNA was subsequently treated with Turbo DNAse I (Ambion) and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1).

Results: The SM RNAseq data identified a number of non-intronic mRNAs that appeard to be bound by the spliceosome. Among these was the BDF2 mRNA, which enocdes for a bromo-domain protein. BDF2 was highly enriched in both SM-IP datasets and was therefore analyzed in more detail. To determine if other non-intronic mRNAs could be regulated by the spliceosome, we analysed the transcriptome in the rrp6Δ, the prp40-1 and a parental strain. Bioinformatic analysis of these data sets revealed that roughly 1% of the non-intronic mRNAs in yeast could be targeted by the spliceosome. TopHat revealed cannonical splice junctions in roughly 30 non-intronic mRNAs, indicating that these messages are spliced.

Conclusions: We demonstrate, for the first time, that the spliceosome can regulate expression of non-intronic mRNAs via one and/or two RNA cleavage events. We refer to this process as Spliceosome Mediated Decay (SMD).

overall_design: We report RNAseq data for two SM immunoprecipitation experiments and RNAseq datasets for the parental strain (BY4741), the prp40-1 mutant, and the rrp6Δ strain.

first: Lidia

last: Vasilieva