title: The Central Role of EED in the Orchestration of Polycomb Group Complexes

city: Atlanta

first: Meng

state: GA

address: 1518 Clifton Rd, #369

department: BIOS

institute: Emory University

summary: Polycomb Repressive Complexes 1 and 2 (PRC1 and 2) play a critical role in the epigenetic regulation of transcription during cellular differentiation, stem cell pluripotency, and neoplastic progression1-3.  Here we show that the Polycomb group protein EED, a core component of PRC2, physically interacts with and functions as part of the PRC1 complex.  Components of PRC1 and PRC2 compete for EED binding.  EED functions to recruit PRC1 to H3K27me3 loci and enhances PRC1 mediated H2A ubiquitin E3 ligase activity.  Taken together, we uncover the integral role of EED as an epigenetic exchange factor coordinating the activities of PRC1 and 2.

overall_design: EED, uH2A, RING1A, RING1B, BMI1 and H3K27Me3 ChIP-seq in EED stable knockdown and control Scramble DU145 prostate cancer cell line

first: Qi

last: Cao

first: Rendong

last: Yang


title: Inhibition of Androgen Receptor and β-catenin activity in prostate cancer

status: Public on Jan 23 2014

city: New York

first: Susan

middle: K.

last: Logan

state: New York

address: 522 1st Ave, Smilow Research Building, Rm # 1211

department: NYU Cancer Institute, Mol Pharm and Biochem

country: USA

institute: NYU School of Medicine

type: Expression profiling by high throughput sequencing

summary: Androgen receptor (AR) is the major therapeutic target in aggressive prostate cancer. However, targeting AR alone can result in drug resistance and disease recurrence. Therefore, simultaneous targeting of multiple pathways could in principle be an effective new approach to treating prostate cancer. Here we provide proof-of-concept that a small molecule inhibitor of nuclear β-catenin activity (called C3) can inhibit both the AR and β-catenin signaling pathways that are often misregulated in prostate cancer. Treatment with C3 ablated prostate cancer cell growth by disruption of both β-catenin/TCF and β-catenin/AR protein interaction, reflecting the fact that TCF and AR have overlapping binding sites on β-catenin. Given that AR interacts with, and is transcriptionally regulated by β-catenin, C3 treatment also resulted in decreased occupancy of β-catenin on the AR promoter and diminished AR and AR/β-catenin target gene expression. Interestingly, C3 treatment resulted in decreased AR binding to target genes accompanied by decreased recruitment of an AR and β-catenin cofactor, CARM1, providing new insight into the unrecognized function of β-catenin in prostate cancer. Importantly, C3 inhibited tumor growth in an in vivo xenograft model, and blocked renewal of bicalutamide-resistant sphere forming cells, indicating the therapeutic potential of this approach.

overall_design: Compare and contrast the expression profile of prostate cancer cells treated with a Wnt inhibitor (C3) with respect to β-catenin and AR knockdown (all samples in duplicates).

first: Eugine

last: Lee

first: Aviv

last: Madar

first: Gregory

last: David

first: Michael

middle: J

last: Garabedian

first: Ramanuj

last: DasGupta

middle: K


title: Large conserved domains of low DNA methylation maintained by 5-hydroxymethycytosine and Dnmt3a (CMS bisulfite-seq)

status: Public on Jan 22 2014

city: Houston

first: Margaret

last: Goodell

state: TX

address: One Baylor Plaza, N1030

institute: Baylor College of Medicine

type: Methylation profiling by high throughput sequencing

summary: Gains and losses in DNA methylation are prominent genomic features of all mammalian cell types.  To gain insight into mechanisms that could promote shifts in DNA 5-hydroxymethylation (5hmc) patterns and thus contribute to cell fate, including malignant transformation, we performed genome-wide mapping of 5hmc in purified wild type and Dnmt3a conditional knockout hematopoietic stem cells (HSCs) using cytosine-5-methylenesulphonate sequencing (CMS-seq). Comparing anti-CMS pulldown to input control, we identified 107,549 and 175,682 peaks of 5hmc enrichment in control and Dnmt3a knockout HSCs, respectively.

overall_design: Whole genome CMS-enriched bisulfite sequencing of secondarily-transplanted wild-type and Dnmt3a conditional knockout hematopoietic stem cells using Illumina HiSeq 2000

first: Benjamin

last: Rodriguez

first: Wei

last: Li


title: Analysis of the recruitment of JARID2 via Xist RNA during XCI by ChIP-seq

city: Zurich

first: Valentina

last: Boeva

address: Universitatstrasse 6

department: Computer Science

country: Switzerland

institute: ETH Zurich

type: Genome binding/occupancy profiling by high throughput sequencing

summary: JARID2 ChIP-seq profile mediated by Xist was investigated using two separate approaches: In the first approach, we use the TX1072 female embryonic stem cell (ESC) line; this is a genetically polymorphic ESC line derived from a mouse with one X-chromosome (X-chr) from the Mus musculus castaneus (Cast) origin and the other of Mus musculus domesticus, C57BL/6  (BL6) strain origin, containing an inducible promoter on the BL6 Xist allele; differentiation and simultaneous induction of Xist for 4 days results in the inactivation of ONLY the BL6-derived X-chr and coating by JARID2 protein. We analysed this ESC line in the undifferentiated state, in which both X-chrs are active (and not coated by JARID2), and also after 4 days of differentiation under Xist inducible conditions, in which cells display one inactive X-chr from BL6 origin and one Xa of the Cast origin; the presence of SNPs allows for the comparison of the degree and localisation of JARID2 enrichment on the inactive X-chr versus the active X-chr; our results suggests that JARID2 is enriched on the inactive X-chr chromosome-wide and not in confined peaks as elsewhere in the genome; This pattern resembles patterns of Xist enrichment published before (Engreitz et al., 2013).  In the second approach, we use a male ESC line that carries an inducible Xist transgene (TG) on chromosome 11 (chr11) (Wutz et al., 2000) in the presence (36:11 WT) or the absence of Eed (36:11 Eed-/-) Induction of Xist TG results in Xist coating and enrichment of JARID2 across the chr11, regardless of the presence of Eed when assess by IF/Xist RNA FISH experiments. We compare inducible and uninducible condition in both the 36:11 WT and Eed-/- ESCs; this way, we could assess the Xist-mediated JARID2 recruitment and the effect of the absence of Eed-/-; Our results shows that Xist induction result in a chromosome-wide enrichment of JARID2 on chr11, in a manner that resembles enrichment on the inactive X-chr; this pattern seem to be independent of EED, showing that in contrast to other regions in the genome where JARID2 occupancy is abrogated in the absence of EED, Xist-mediated JARID2 recruitment is not affected.

overall_design: JARID2 ChIP-seq analysis in differentiating female ESCs (TX1072) and in a male ESC harbouring a Xist transgene on chromosome 11 in WT and Eed-/- genetics settings

first: Simao

middle: T

last: da Rocha

first: Edith

last: Heard


title: SM014 - SARS MA15 wild type, and SARS nsp16 mutant virus infections of C57BL6 mice - A time course

city: Seattle

last: Katze

state: WA

address: Rosen Building 960 Republican St.

department: Microbiology

institute: University of Washington

summary: This purpose of this experiment was to investigate the transcriptional differences between mice infected with SARS MA15 or SARS nsp16 mutant viruses.

overall_design: Overview of Experiment: Groups of 20 week old C57BL6 mice were infected with wild type SARS MA15 or SARS nsp16 mutant viruses.  Infections were done at 10^5 PFU or time-matched mock infected.  Time points were 1, 2, 4 and 7 d.p.i.  There were 3-4 animals/dose/time point.  Lung samples were collected for virus load, transcriptional and proteomics analysis. Weight loss and animal survival were also monitored.

first: Ralph

last: Baric

first: Lynn

last: Law

first: Vineet

last: Menachery

first: Jean

last: Chang


title: Type I Interferon induces binding of STAT1 to Bcl6: Divergent Roles of STAT-family transcription factors in the T follicular helper cell genetic program

city: Bethesda

first: Golnaz

last: Vahedi

state: MD

address: 9000 Rockville Pike Bldg 10 Rm 13C101A

department: NIAMS

institute: National Institutes of Health

summary: CD4+ T follicular helper cells (TFH) are critical for the formation and function of B cell responses to infection or immunization, but also play an important role in autoimmunity. The factors that contribute to the differentiation of this helper cell subset are incompletely understood, although several cytokines including IL-6, IL-21 and IL-12 can promote TFH cell formation. Yet, none of these factors, nor their downstream cognate STATs, have emerged as non-redundant, essential drivers of TFH cells.  This suggests a model in which multiple factors can contribute to the phenotypic characteristics of TFH cells. As type I interferons (IFNs) are often generated in immune responses, we set out to investigate if these factors are relevant to TFH cell differentiation. Type I IFNs promote Th1 responses, thus one possibility was these factors antagonized TFH-expressed genes.  However, we show that type I IFNs (IFN-α/β) induced Bcl6 expression, the master regulator transcription factor for TFH cells, and CXCR5 and PD-1 (encoded by Pdcd1), key surface molecules expressed by TFH cells. In contrast, type I IFNs failed to induce IL-21, the signature cytokine for TFH cells. The induction of Bcl6 was regulated directly by STAT1, which bound to the Bcl6, Cxcr5 and Pdcd1 loci. These data suggest that type I IFNs (IFN-α/β) and STAT1 can contribute to some features of TFH cells but are inadequate in inducing complete programming of this subset.

overall_design: The role of STAT1 in type I interferon treated CD4+ T cells was investigated by Chip-seq of STAT1.

first: Amanda

middle: C

last: Poholek

first: Shingo

last: Nakayamada

first: Yuka

last: Kanno

first: Hong-wei

last: Sun

first: John

last: O'Shea


title: WTAP is a novel oncogenic protein in Acute Myeloid Leukemia

city: Los Angeles

first: Philip

middle: James

last: Uren

state: CA

address: 1050 Childs Way, RRI201

institute: University of Southern California

summary: Acute myeloid leukemia (AML) continues to have the lowest survival rates of all leukemias. Therefore, new therapeutic strategies are urgently needed to improve clinical outcomes for AML patients. Here, we report a novel role for Wilms’ tumor 1-associated protein (WTAP) in pathogenesis of AML. We have performed RNA-Seq in K562 cells with knockdown of WTAP to ascertain which genes it regulates.

overall_design: We have 2 replicates of total RNA for K562 cells and 2 replicates with WTAP knocked down

first: Luiz

middle: O

last: Penalva


title: Gonadal DNA methylomes of half-smooth tongue sole Cynoglossus semilaevis

city: Shenzhen

first: Hong

middle: zhi

last: Cao

state: Guangdong

address: F5, Main Building, Beishan Industrial Zone, Beishan Road, Yantian District

department: Bioinformatics

country: China

institute: BGI-SHENZHEN

summary: Environmental sex determination (ESD) occurs in divergent, phylogenetically unrelated taxa, and in some species co-occurs with genetic sex determination (GSD) mechanisms. Although epigenetic regulation in response to environmental effects has long been proposed to be associated with ESD, a systemic analysis on epigenetic regulation of ESD is still lacking. Using half-smooth tongue sole (Cynoglossus semilaevis) as a model – a marine fish which has both ZW chromosomal GSD and temperature-dependent ESD – we investigated the role of DNA methylation in transition from GSD to ESD by comparing gonadal DNA methylomes of parental females, parental pseudo-males, F1 females, F1 pseudo-males and normal males.

overall_design:
To assess the gonadal DNA methylome patterns across different sexual types of tongue sole, we carried out BS-seq on bisulfite converted DNA extracted from adult gonads of parental females, parental pseudo-males, and F1 pseudo-males and females from a cross between a parental pseudo-male and a normal female. We also sampled normal male individuals as a control for the normal male DNA methylation pattern. For each of the five samples, two biological replicates were utilized, with each replicate being pooled by five fish. The phenotype and genotype of each selected fish was identified by the histological analysis and PCR validation using the W chromosome specific marker.

DNA were isolated from five pooled gonads of the same replicate, then 5 μg DNA was used to do the bisulfite conversion and BS-seq. The bisulfite conversion of sample DNA was carried out using a modified NH4HSO3-based protocol (Hayatsu et al. 2006). The paired-end library construction and sequencing were carried out using Illumina HiSeq 2000, according to the manufacturer’s instructions (Illumina). We also mixed 25 ng cl857 Sam7 Lambda DNA in each sample to use as conversion quality control for each library.

first: Changwei

last: Shao

first: Qiye

last: Li

first: Songlin

last: Chen

first: Pei

last: Zhang

first: Jinmin

last: Lian

first: Qiaomu

last: Hu

first: Bing

first: Lijun

last: Jin

first: Shanshan

last: Liu

first: Zongji

last: Wang

first: Hongmei

last: Zhao

first: Zonghui

first: Zhuo

last: Liang

first: Yangzhen

first: Qiumei

last: Zheng

first: Yong

first: Jun

first: Guojie


title: RNA-Seq and RNA Polymerase II ChIP-Seq of mouse spermatogenesis

first: Gennady

last: Margolin

address: 9000 Rockville Pike

summary: To characterize gene expression in spermatogenesis we sequenced eight mRNA samples from testes of juvenile mice aged between 6 and 38 days post partum. We then classified gene expression profiles and found over 1,000 meiotically-expressed protein-coding genes that have not been previously reported in relation to spermatogenesis. Furthermore, we developed an in silico de-convolution approach to estimate cell type-specific gene expression from temporal gene expression profiles and applied it to our dataset. To further characterize transcription during spermatogenesis, we determined RNA polymerase II (RNAPII) distribution along the genome using ChIP-Seq.

overall_design: mRNA sequencing at 8 time points during spermatogenesis was used to characterize differential gene expression; RNA Pol II ChIP-Seq at two time points was used to estimate RNA Pol II accumulation at promoters.

first: Pavel

middle: P

last: Khil

first: Joongbaek

last: Kim

first: Marina

middle: A

last: Bellani

first: Rafael

middle: D

last: Camerini-Otero


title: Runx3 Regulates Interleukin-15-Dependent Natural Killer Cell Activation

city: Rehovot

first: Joseph

last: Lotem

department: Molecular genetics

country: Israel

institute: Weizmann institute of science

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

overall_design: Refer to individual Series


title: Hypermethylation in the ZBTB20 gene is associated with major depressive disorder

status: Public on Jan 21 2014

city: London

first: Matthew

middle: N

last: Davies

state: Greater London

address: St Thomas' Hospital Campus

department: Twin Research

country: United Kingdom

institute: King's College London

summary: Genome-wide MeDIP-Sequencing was carried out on a total of 50 monozygotic twin pairs from the UK and Australia that are discordant for depression. We show that major depressive disorder is associated with significant hypermethylation within the coding region of ZBTB20, and is replicated in an independent cohort of 356 unrelated case-control individuals. The twins with major depressive disorder also show increased global variation in methylation in comparison with their unaffected co-twins. ZBTB20 plays an essential role in the specification of the Cornu Ammonis-1 field identity in the developing hippocampus, a region previously implicated in the development of major depressive disorder.

overall_design: Epigenetic study of MZ twins discordant for Major Depressive Disorder

first: Jordana

last: Bell


title: Gene expression in feather dermal papilla (DP)

city: Fuzhou

first: Yue

last: Zhicao

state: Fujian

address: Institute of Life Sciences, Fuzhou University

department: Institute of Life Sciences

institute: Fuzhou University

summary:
The feather follicle is a “professional” regenerative organ that undergoes natural cycling and, regeneration after wound plucking. Similar to mammalian hair follicle, dermal papilla (DP) controls feather regeneration, shape, size, and axis. Here we report gene expression profiling for feather DP at different growth stages.
For growth phase, we compared gene expression of DP, the ramogenic zone of feather branching epithelium (Erz) and the mesenchymal pulp (Pp). We also compared gene expression of DP at resting phase. To characterize the feather regeneration process, we further profiled gene expression at Day-2 and Day-4 post wound. Our results provide a resource for investigating feather growth and regeneration.

overall_design: Examination of gene expression in dermal papilla (DP) at growth phase and resting phase feather follicle, and during feather regeneration.

first: Zhicao

last: Yue

first: Hao

last: Han


title: Anp32e is a histone chaperone that removes H2A.Z from chromatin

city: ILLKIRCH-GRAFFENSTADEN

first: Christophe

last: Papin

address: 1 rue Laurent Fries

country: France

institute: IGBMC - CNRS UMR 7104 - Inserm U 964

summary: We report a genome wide enrichment, redistribution and accumulation of H2A.Z at specific chromatin control regions, in particular at enhancers and insulators, in mouse embryonic fibroblasts depleted for Anp32e (MEFs Anp32e-/-).

overall_design: H2A.Z ChIP-seq in MEFs WT (+/+) or KO (-/-) for Anp32e.

first: Arnaud

last: Obri

first: Khalid

last: Ouararhni

first: Marie-Laure

last: Diebold

first: Kiran

last: Padmanabhan

first: Martin

last: Marek

first: Isabelle

last: Stoll

first: Ludovic

last: Roy

first: Patrick T.

last: Reilly

first: Tak W.

last: Mak

first: Stefan

last: Dimitrov

last: Romier

first: Ali

last: Hamiche


title: Conservation and divergence of regulatory strategies and the origin of tetrapod digits

city: Lausanne

first: Marion

last: LELEU

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

department: School of Life Sciences

institute: EPFL

summary: The evolution of tetrapod limbs from fish fins enabled the conquest of land by vertebrates and thus represents a key step in evolution. Despite the use of comparative gene expression analyses, critical aspects of this transformation remain controversial, in particularly the origin of digits. Hoxa and Hoxd genes are essential for the specification of the different limb segments and their functional abrogation leads to large truncations of the appendages. Here we show that the selective transcription of mouse Hoxa genes in proximal and distal limbs is related to a bimodal higher order chromatin structure, similar to that reported for Hoxd genes, thus revealing a generic regulatory strategy implemented by both gene clusters during limb development. We found the same bimodal chromatin architecture in fish embryos, indicating that the regulatory strategy used to pattern tetrapod limbs predates the divergence between fish and tetrapods. However, when assessed in mice, both fish regulatory domains triggered transcription in proximal, rather than distal limb territories, supporting an evolutionary scenario whereby digits arose as true tetrapod novelties through genetic retrofitting of a preexisting bimodal chromatin framework. We discuss the possibility to consider regulatory circuitries, rather than expression patterns, as essential parameters to define evolutionary synapomorphies.

sample_organism:
Danio rerio
Mus musculus

platform_organism:
Danio rerio
Mus musculus

overall_design: Circular Chromosome Conformation Capture (4C seq) at the mouse HoxA and HoxD loci in proximal and distal forelimbs and forebrain at E12.5 and at the zebrafish HoxAa, HoxAb and HoxDa loci in 5 dpf whole embryos.

first: J

middle: M

last: Woltering

first: D

last: Noordermeer

first: M

last: Leleu

last: Duboule


title: Androgen receptor uses relaxed response element stringency for selective chromatin binding and transcriptional regulation in vivo

city: Helsinki

first: Olli

middle: A.

last: Jänne

department: Inst. of Biomedicine/Physiology

country: Finland

institute: University of Helsinki

summary: We report the in vivo androgen receptor (AR) binding sites in caput epididymis of intact SPARKI and wild-type mice using ChIP-sequencing. SPARKI (specificity affecting androgen receptor knock-in) mouse line has the second zinc finger of AR replaced by that of glucocorticoid receptors. In vivo analysis of SPARKI and wild-type AR genome-wide binding sites identified cis-element with less stringent sequence requirements that specify selective AR-binding sites. The ChIP experiments have been performed using antibody specific to AR and IgG non-specific antibody as a negative control.

overall_design: Examination of AR binding sites in epididymis of wild-type and SPARKI mice using ChIP-seq. Two biological replicates from intact wild-type and SPARKI mice and two control IgG samples were sequenced and used in peak calling. To increase the depth of the analysis, replicate ChIP-seq samples were merged and the concatanated samples were used in peak calling.

first: Biswajyoti

last: Sahu

first: Päivi

last: Pihlajamaa

first: Vanessa

last: Dubois

first: Stefanie

last: Kerkhofs

first: Frank

last: Claessens


title: Next generation sequencing of the transcriptome in MCF-7 cells with/without SRA knockdown

city: Boulder

first: Linghe

last: Xi

state: CO

address: 3415 Colorado Avenue

institute: CU Boulder

summary: We employed next generation sequencing to examine whether knocking down the steroid receptor RNA activator (SRA) gene significantly affect the expression levels of certain genes in MCF-7 cells. MCF-7 cells were transfected with either a pool of four non-target control siRNAs or a pool of four SRA siRNAs for 32 hrs. 157 million reads were generated from triplicate samples of the control group; 151 million reads were generated from triplicate samples of the SRA knockdown group. Six genes were identified as significantly changed in the expression levels with the cutoff of q value ≤ 0.05, fold change ≤ 0.5 or ≥ 2, and reads per kilobase per million mapped reads (RPKM) ≥ 1. However, except for SRA itself, the other five genes were shown by real-time PCR to be only affected by one siRNA in the SRA siRNA pool. Further analysis of this dataset with different cuttoff setting may reveal true SRA-regulated genes in MCF-7.

overall_design: MCF-7 cells were cultured in high glucose DMEM with 10% fetal bovine serum, 2 mM Glutamax-1, 100 units/ml penicillin and 100 μg/ml streptomycin. ON-TARGETplus SMARTpool for human SRA (Thermo Scientific, L-027192-00-0005) was used to knockdown SRA (siSRA) and ON-TARGETplus Non-targeting Pool Thermo Scientific, D-001810-10-05) was used as a negative control (siCtrl). A total of 25 nM siRNA was transfected in 6-well dishes using Lipofectamine™ RNAiMAX Reagent (Life Technologies, Invitrogen) following the manufacturer’s recommendations. Polyadenylated RNA was purified from the cells 32 hrs after transfection. cDNA libraries were prepared and double-stranded cDNA was fragmented using DNase I according to Illumina specifications, prior to adaptor ligation. Sequencing libraries were amplified and sequenced using an Illumina HiSeq 2000 sequencer.

first: Thomas

middle: R

last: Cech


title: Transcriptome Analysis of T3SS1 in Vibrio parahaemolyticus

city: Pullman

first: Douglas

middle: Ruben

last: Call

state: Washington

address: 240 SE Ott Rd

department: Paul G. Allen School for Global Animal Health

institute: Washington State University

summary: Comparative transcriptional mRNA profiles were generated of bacterial pathogen Vibrio parahaemolyticus under conditions that induce activity of virulence factor type III secretion system 1 (T3SS1).  Induction conditions included growth of the bacteria in Dulbecco's Modified Eagle Medium (DMEM), overexpression of transcriptional activator exsA and contact with HeLa cells in Hank's Balanced Salt Solution (HBSS), while non-inducing conditions included growth in Luria-Bertani medium supplemented with 2.5% (w/v) NaCl (LB-S) and overexpression of transcriptional anti-activator exsD.  Transcriptome profiles of induction conditions were cross-compared against background non-inducing conditions and time points during HeLa cell infection (2 hr, 3 hr, 4 hr, 6 hr, 8 hr) were compared against pre-infection (0 hr) to identify genes important in T3SS1 activity and pathogenesis.

overall_design: Duplicate mRNA transcriptome profiles of V. parahaemolyticus grown in T3SS1 inducing (DMEM, exsA expression) and non-inducing (LB-S, exsD expression) conditions as well as during HeLa cell infection (0 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr) were generated by deep sequencing using an Illumina MiSeq

first: Seth

last: Nydam


title: High throughput screening for chemical inducers of stumpy formation in Trypanosoma brucei

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

institute: Centre for Immunity, Infection and Evolution

summary: During the bloodstream stage of the Trypanosoma brucei lifecycle, the parasite exists as the proliferative slender-form or the non-proliferative, transmissible, stumpy-form. The transition from the slender to stumpy-form is stimulated by a density-dependent mechanism and is important in infection dynamics, ordered antigenic variation and disease transmissibility. Here, we use a monomorphic reporter cell line in a whole-cell fluorescence-based assay to screen over 6000 small molecules from a kinase-focussed compound library for their ability to induce stumpy-like formation in a high-throughput screening programme. This identified one compound able to induce modest, yet specific, changes in gene expression indicative of a partial differentiation to stumpy forms. This not only provides a potential tool for the further understanding of stumpy formation, but also demonstrates the use of high throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.

overall_design: Examination of gene expression in response to treatment with DDD00015314.

first: Paula

last: MacGregor

first: Keith

last: Matthews


title: Genomic features and regulatory roles of intermediate-size non-coding RNAs in Arabidopsis

status: Public on Jan 20 2014

city: Beijing

first: Xuncheng

last: Wang

address: 5 Yiheyuan Road

institute: Peiking University

type: Non-coding RNA profiling by high throughput sequencing

summary: Recent advances in genome-wide techniques allowed the identification of thousands of non-coding RNAs with various sizes in eukaryotes, some of which have further been shown to serve important functions in many biological processes. However, in model plant Arabidopsis, novel intermediate-sized ncRNAs (im-ncRNAs) (50~300nt) have very limited information. By using a modified isolation strategy combined with deep-sequencing technology, we identified 838 im-ncRNAs in Arabidopsis globally. More than half (58%) are new ncRNA species, mostly evolutionary divergent. Interestingly, annotated protein-coding genes with 5’-UTR derived novel im-ncRNAs tend to be highly expressed. For intergenic im-ncRNAs, their average abundances were comparable to mRNAs in seedlings, but subsets exhibited significantly lower expression in senescing leaves. Further, intergenic im-ncRNAs were regulated by similar genetic and epigenetic mechanisms as those of protein-coding genes, and some showed developmentally-regulated expression patterns. Large-scale reverse genetic screening showed that the down-regulation of a number of im-ncRNAs resulted in either obvious molecular changes or abnormal developmental phenotypes in vivo, indicating the functional importance of im-ncRNAs in plant growth and development. Together, our results demonstrate that novel Arabidopsis im-ncRNAs are developmentally-regulated and functional components discovered in the transcriptome.

overall_design: Genome-wide maps of Intermediate-size Non-coding RNAs in Arabidopsis

first: Yuqiu

first: Danmeng

last: Zhu

first: Xingwang

last: Deng


title: The dsRBP and inactive editor, ADR-1, utilizes dsRNA binding to regulate A-to-I RNA editing across the C. elegans transcriptome

city: La Jolla

first: Boyko

last: Kakaradov

address: 2880 Torrey Pines Scenic Dr.

institute: UCSD

summary:
Purpose: The purpose of this experiment is to expand the repertoire of C. elegans edited transcripts and identify the roles of ADR-1 as indirect regulator of editing and ADR-2 as the only active deaminase in vivo.
Methods: Strand-specific RNA sequencing of wild-type and adr mutant worms, followed by a novel RNA variant calling and comparative analysis pipeline.
Results: Despite lacking deaminase function, ADR-1 affects editing of over 60 adenosines within the 3’ UTRs of 16 different mRNAs. Furthermore, ADR-1 interacts directly with ADR-2 substrates, even in the absence of ADR-2; and mutations within its dsRNA binding domains abolished both binding and editing regulation.
Conclusions: ADR-1 acts as a major regulator of editing by binding ADR-2 substrates in vivo and raises the possibility that other dsRNA binding proteins, including the inactive human ADARs, regulate RNA editing by deaminase-independent mechanisms.

first: Michael

last: Washburn

first: Balaji

last: Sundararaman

first: Emily

last: Wheeler

first: Shawn

last: Hoon

first: Gene

middle: W

last: Yeo

first: Heather

last: Hundley


title: DNase I hypersensitivity and algorithmic prediction of TF binding in early pancreatic mES directed differentiation

status: Public on Jan 19 2014

city: Cambridge

first: David

middle: K.

last: Gifford

state: MA

address: 32 Vassar Street

department: Electrical Engineering and Computer Science

institute: Massachusetts Institute of Technology

summary: This dataset uses DNase-seq to profile the genome-wide DNase I hypersensitivity of mES and mES-derived cells along an early pancreatic lineage and provides the locations of putative Transcription Factor (TF) binding sites using the PIQ algorithm. DNase-seq takes advantage of the preferential cutting of DNase I in open chromatin and steric blockage of of DNase I by tightly bound TFs that protect associated genomic DNA sequences. After deep sequencing of DNase I–digested genomic DNA from intact nuclei, genome-wide data on chromatin accessibility as well as TF-specific DNase I protection profiles that reveal the genomic binding locations of a majority of TFs are obtained. Such TF signature ‘DNase profiles’ reflect the effect of the TF on DNA shape and local chromatin architecture, extending hundreds of base pairs from a TF binding site, and these profiles are centered on ‘DNase footprints’ at the binding motif itself, which reflects the biophysics of protein-DNA binding. An algorithm, PIQ, is then applied that models the specific profile of each factor, and in combination with sequence information predicts the likely binding locations of over 700 TFs genome wide.

overall_design: This dataset includes DNase-seq hypersensitivity data from 6 mES-derived cell types: mESC, Mesendoderm, Mesoderm, Endoderm, Intestinal Endoderm, and Prepancreatic Endoderm. For each cell type, TF binding site predictions are made based on the identification TF-specific DNase-seq profiles over any of 1331 possible binding motifs. After significance thesholding, genome-wide binding site predictions for <700 TFs are included.

first: Richard

middle: I

last: Sherwood

first: Tatsunori

last: Hashimoto

first: Charles

last: O'Donnell

first: Sophia

last: Lewis

first: Amira

last: Barkal

first: John

last: van Hoff

first: Vivek

last: Karun

first: Tommi

last: Jaakkola


title: Tissue-specific pioneer factors associate with androgen receptor cistromes and transcription programs

first: Olli

last: Jänne


title: Genome-wide maps of Runx3 bound regions in splenic NK cells activated in vivo with IL-15

first: Joseph

last: Lotem

summary: ChIP-seq was conducted using freshly isolated splenic WT NK cells from IL-15/Ra treated mice with anti-Runx3 antibody (Ab) and non-immune serum (NIS) as control.

overall_design: Runx3 and NIS IP from splenic NK cells of IL-15/Ra treated WT mice, isolated by negative selection using NK cell isolation kit (R&D) followed by sorting of NKp46+ cells.

first: Ditsa

last: Levanon

first: Dena

last: Leshkowitz

first: Yoram

last: Groner


title: The evolution of lncRNA repertoires and expression patterns in tetrapods

city: Lyon

first: Anamaria

last: Necsulea

address: 43 Bd. du 11 Novembre 1918

department: Laboratoire de Biométrie et Biologie Evolutive

institute: CNRS, Université Claude Bernard Lyon 1

summary: Only a minuscule fraction of long non-coding RNAs (lncRNAs) are well characterized. The evolutionary history of lncRNAs can provide insights into their functionality, but comparative analyses have been precluded by our ignorance of lncRNAs in non-model organisms. Here, we use RNA sequencing to identify lncRNAs in eleven tetrapod species and we present the first large-scale evolutionary study of lncRNA repertoires and expression patterns. We identify ~11,000 primate- specific lncRNA families, which show evidence for selective constraint during recent evolution, and ~2,400 highly conserved lncRNAs (including ~400 genes that likely originated more than 300 million years ago). We find that lncRNAs, in particular ancient ones, are generally actively regulated and may predominantly function in embryonic development. lncRNA X-inactivation patterns reveal an extremely female-biased monotreme-specific lncRNA, which may partially compensate X-dosage in this lineage. Most lncRNAs evolve rapidly in terms of sequence and expression levels, but global patterns like tissue specificities are often conserved. We compared expression patterns of homologous lncRNA and protein-coding families across tetrapods to reconstruct an evolutionarily conserved co-expression network. This network, which surprisingly contains many lncRNA hubs, suggests potential functions for lncRNAs in fundamental processes like spermatogenesis or synaptic transmission, but also in more specific mechanisms such as placenta growth suppression through miRNA production.

sample_organism:
Xenopus tropicalis
Gallus gallus
Ornithorhynchus anatinus
Macaca mulatta
Gorilla gorilla
Homo sapiens
Mus musculus
Monodelphis domestica

overall_design: [Batch 1 and 2] To broaden our understanding of lncRNA evolution, we used an extensive RNA-seq dataset to establish lncRNA repertoires and homologous gene families in 11 tetrapod species. We analyzed the poly- adenylated transcriptomes of 8 organs (cortex/whole brain without cerebellum, cerebellum, heart, kidney, liver, placenta, ovary and testis) and 11 species (human, chimpanzee, bonobo, gorilla, orangutan, macaque, mouse, opossum, platypus, chicken and the frog Xenopus tropicalis), which shared a common ancestor ~370 millions of years (MY) ago. Our dataset included 47 strand-specific samples, which allowed us to confirm the orientation of gene predictions and to address the evolution of sense-antisense transcripts. See also GSE43721 (Soumillon et al, Cell Reports, 2013) for three strand-specific samples for mouse brain, liver and testis.

first: Magali

last: Soumillon

first: Angélica

last: Liechti

first: Tasman

last: Daish

first: Ulrich

last: Zeller

first: Julie

last: Baker

first: Frank

last: Grutzner

first: Henrik

last: Kaessmann

first: Maria

last: Warnefors


title: Tissue-specific pioneer factors associate with androgen receptor cistromes and transcription programs. [ChIP-Seq]

summary: We report the in vivo androgen receptor (AR) binding sites in murine prostate, epididymis and kidney in response to physiological androgen testosterone using ChIP-sequencing and gene expression profiling by microarray. From AR cistrome analysis, we identified tissue-specific collaborating factors i.e. FoxA1 in prostate, Hnf4a in kidney and AP2a in epididymis and validated by ChIP-seq. The ChIP experiments have been performed using antibodies specific to AR, FoxA1, Hnf4a, AP-2a and IgG non-specific antibody as a negative control.

overall_design: Examination of AR binding sites in murine androgen-responsive tissues prostate, epididymis and kidney using ChIP-seq. Further analysis of AR cistromes led to identification of tissue-specific collaborating factors and these collaborating factors are validated by ChIP-seq from the same tissues. Two parallel IgG samples were sequenced, merged together and used as a control data set. Parallel ChIP-seq samples were sequenced and merged for each replicate wherever required to contain approximately the same amount of reads across all tissues and conditions. All ChIP-seq experiments are performed in biological duplicates except for the castrated conditions.

first: Päivi

last: Pihlajamaa

first: Biswajyoti

last: Sahu

first: Lauri

last: Lyly

first: Viljami

last: Aittomäki

first: Sampsa

last: Hautaniemi


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and fl4 mutant Transcriptomes

status: Public on Jan 18 2014

city: Shanghai

first: Weiwei

last: Qi

address: 333 Nanchen Road

institute: Shanghai University

summary: Analysis of  gene expression level. The hypothesis tested in the present study was that fl4 mutant influence the ER stress pathway. Results provide important information of the gene expression level regulation of different ER stress pathways and apoptosis

overall_design: Endosperm mRNA profiles of 19DAP wild type (WT) and fl4 mutant were generated by deep sequencing, in triplicate, using Illumina Hiseq 2000 (Zea mays).

first: Guan

last: Wang


title: Next Generation Sequencing Facilitates Quantitative Analysis of effect of knockdown of GATA2 on AR binding sites

city: Columbus

first: Qianben

state: Ohio

address: 460 W 12th Avenue

department: Molecular and Cellular Biochemistry and the Comprehensive Cancer Center

institute: Ohio State University

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare AR binding activity in LNCaP cells with and without knockdown of GATA2.
Methods: LNCaP cells between passage number 32-34 were used for assay. Cells are transfected with GATA2 specific or nonspecific siRNA and ChIP was performed, the ChIP producted was further used to generate library with illumina ChIP-seq kit. Hi-seq 2500 was used for sequencing and the data was analyzed by MACs for peaks.
Results: GATA2 knockdown lead to changes of AR binding activity , in most AR binding sites, AR shows decreased bindig activity. Only small percent sites show increased binding.
Conclusions: Our study represents the first detailed analysis of the relationship between GATA2 and AR binding in whole genomic DNA.These results demostrate GATA2 play a critical role in AR activity in prostate cancer.

overall_design: LNCaP cells was used as cell model were treated with specific GATA2 siRNA.Library was sequenced using Illumina HI-seq 2500.

first: Dayong

last: Wu

first: Xiangtal

last: Liu


title: Serum Response Factor binding in Dstn-corn1 mutant and WT murine cornea

city: Madison

first: Sakae

last: Ikeda

state: WI

address: 425G Henry Mall

department: Medical Genetics

institute: University of Wisconsin - Madison

summary: We report the application of chromatin immunoprecipitation sequencing to identify binding sites of the transcription factor serum response factor (SRF) in the cornea of WT and Dstn-corn1 mutant mice

overall_design: Examination of SRF binding sites in WT and Dstn-corn1 mutant cornea

first: Sharolyn

middle: V

last: Kawakami


title: Transcriptional profiling in dioecious plant Populus cathayana reveals potential and sex-specific molecular adaptations to solar UV-B radiation.

city: Chengdu

first: HAO

last: JIANG

address: RENMING ROAD SOUTH

institute: IMDE-CAS

summary: Recently, we found a dioecious plant Populus cathayana males possess a greater tolerance to enhanced UV-B radiation than do females. To carry this work forward, comparative transcriptome analyses were carried out. Similar to previous studies, a set of conserved functions and pathways related to UV-B stress were detected in males and females, regardless of the sex. In addition, sex-specific responses via transcriptome remodeling were also detected as shown in the changes of sex-related gene expression occurred in some pathways. For example, a lot of differentially expressed genes (DEGs) involved in amino acid metabolism were mainly up-regulated in males, but down-regulated in females. Moreover, we found some DEGs expressed predominantly or exclusively in one sex, which may directly contribute to sex-related physiological responses.

overall_design: 4 samples examined: (i) males exposure to decreased solar UV-B radiation (MC); (ii) females exposure to decreased solar UV-B radiation (FC); (iii) males exposure to ambient solar UV-B radiation (MU); and (iv) females exposure to ambient solar UV-B radiation (FU). Nine plants of each sex were exposed to each treatment, and RNA samples from the 9 individuals were pooled with equal proportion.

first: Jiang

last: Hao

first: Zhang

last: Sheng

first: Feng

last: Lihua

first: Korpelainen

last: Helena

first: Li

last: chunyang


title: T-cell factor 4 and β-catenin chromatin occupancies pattern zonal liver metabolism.

city: Villeurbanne

first: Philippe

last: Veber

address: 16 rue Raphael Dubois

institute: CNRS

summary: β-catenin signaling can be both a physiological and an oncogenic pathway in the liver. It controls compartmentalized gene expression, allowing the liver to ensure its essential metabolic function. It is activated by mutations in 20 to 40% of hepatocellular carcinomas with specific metabolic features. We decipher the molecular determinants of β-catenin-dependent zonal transcription using mice with β-catenin-activated or -inactivated hepatocytes, characterizing in vivo their chromatin occupancy by Tcf4 and β-catenin, their transcriptome and their metabolome. We find that Tcf4 DNA-bindings depend on β-catenin. Tcf4/β-catenin binds Wnt-responsive elements preferentially around β-catenin-induced genes. In contrast, genes repressed by β-catenin bind Tcf4 on Hnf4-responsive elements. β-catenin, Tcf4 and Hnf4α interact, dictating β-catenin transcription which is antagonistic to that elicited by Hnf4α. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by β-catenin, partly through xenobiotic nuclear receptors. We conclude that β-catenin patterns the zonal liver together with Tcf4, Hnf4α and xenobiotic nuclear receptors. This network represses lipid metabolism, and exacerbates glutamine, drug and bile metabolism, mirroring hepatocellular carcinomas with β-catenin mutational activation.

overall_design: In vivo liver samples in 4 conditions: Betacat activated  (WCE, Tcf4 chipseq, Betacat chipseq, mRNAseq with 2 replicates), Betacat null (WCE, Tcf4 chipseq, mRNAseq with 2 replicates), Betacat control (mRNAseq with 2 replicates), Wild type (mRNAseq with 2 replicates)

first: Angélique

last: Gougelet

first: Cyril

last: Torre

first: Chiara

last: Sartor

first: Laura

last: Bachelot

first: Pierre-Damien

last: Denechaud

first: Cécile

last: Godard

first: Marthe

last: Moldes

first: Anne-Françoise

last: Burnol

first: Céline

last: Dubuquoy

first: Benoit

last: Terris

first: François

last: Guillonneau

first: Tao

last: Ye

first: Michael

last: Schwarz

first: Albert

last: Braeuning

first: Christine

last: Perret

first: Sabine

last: Colnot


title: Mll2 is required for H3K4 trimethylation on bivalent promoters in ES cells whereas Mll1 is redundant

city: Nijmegen

first: Hendrik

last: Marks

address: Geert Grooteplein 26/28

department: Molecular Biology

country: Netherlands

institute: Radboud University Nijmegen, RIMLS

summary: Trimethylation of histone 3 lysine 4 (H3K4me3) at promoters of actively transcribed genes is a universal epigenetic mark and a key product of Trithorax-Group action. Here we show that Mll2, one of the six Set1/Trithorax-type H3K4 methyltransferases in mammals, is required for trimethylation of bivalent promoters in mouse embryonic stem cells. Mll2 is bound to bivalent promoters but also to most active promoters, which do not require Mll2 for H3K4me3 or mRNA expression. In contrast, the Set1 complex (Set1C) subunit Cxxc1 is primarily bound to active but not bivalent promoters. This indicates that bivalent promoters rely on Mll2 for H3K4me3 whereas active promoters have more than one bound H3K4 methyltransferase including Set1C. Removal of Mll1, sister to Mll2, had almost no effect on any promoter unless Mll2 was also removed indicating functional back-up between these enzymes. Except for a subset, loss of H3K4me3 on bivalent promoters did not prevent responsiveness to retinoic acid thereby arguing against a priming model for bivalency. In contrast, we propose that Mll2 is the pioneer trimethyltransferase for promoter definition in the naïve epigenome and Polycomb-Group action on bivalent promoters blocks premature establishment of active, Set1C bound, promoters.

overall_design: ChIP-Seq to study MLL2 function using H3K4me3 (12 samples), H3K27me3 (4 samples), Pol2 (1 sample) or GFP (7 samples) antibody, and 6 RNA-Seq profiles

first: Sergei

last: Denissov

first: Helmut

last: Hofemeister

first: Andrea

last: Kranz

first: Giovanni

last: Ciotta

first: Sukdeep

last: Singh

first: Konstantinos

last: Anastassiadis

middle: G

last: Stunnenberg

first: A

middle: F

last: Stewart


title: MARIS: Method for Analyzing RNA following Intracellular Sorting [RNA-seq]

status: Public on Jan 17 2014

city: Boston

first: Sinisa

last: Hrvatin

address: 220 Longwood ave, GB405

department: Neurobiology

institute: Harvard Medical School

summary: We've developed a new Method to Analyze RNA following Intracellular Sorting (MARIS) allowing us to carry out gene expression studies on cells sorted based on intracellular immunoflourescence. The purpose of this study is to determine the degree of bias that MARIS introduces on gene expression. We report RNA-seq gene expression data from human embryonic stem cells differentiated to a stage in which insulin-expressing cells are present. Gene expression data using RNA isolated from live cells is compared to gene expression data using RNA isolated from MARIS processed cells (fixed, permeabilized, antibody stained and mock sorted) to determine the degree of correlation in gene expression between these two biologically identical samples.

overall_design: Human embryonic stem cells are differentiated to a stage in which insulin-expressing cells are present and split into two biologically identical samples. RNA is immediately isolated from one sample using the RNeasy protocol (live sample). RNA is isolated from the second sample following MARIS (processed sample) with all cells collected after the sort in order to keep the cell type composition between the live and processed samples the same.

first: Siniša

first: Francis

last: Deng

first: Charles

last: O'Donnell

first: David

middle: K

last: Gifford

first: Douglas

last: Melton


title: MARIS: Method for Analyzing RNA following Intracellular Sorting

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

overall_design: Refer to individual Series


title: Temporal patterns of gene expression in developing maize endosperm identified through transcriptome sequencing

city: Tucson

first: Ramin

last: Yadegari

state: Arizona

address: 1140 E. South Campus Dr.

department: Plant Sciences

institute: University of Arizona

summary: Endosperm is a filial structure resulting from a second fertilization event in angiosperms. As an absorptive storage organ, endosperm plays an essential role in support of embryo development or seedling germination. The accumulation of carbohydrate and protein storage products in cereal endosperm provides humanity with a major portion of its food, feed and renewal resources. However, little is known regarding the regulatory gene networks controlling endosperm proliferation and differentiation. As a first step towards understanding these processes, we have profiled all mRNAs in kernel and endosperm of maize at eight successive stages during the first 12 days after pollination. Analysis of these gene sets has identified temporal programs of gene expression including hundreds of transcription-factor genes. We also show a close correlation of these sequentially expressed gene sets with distinct spatial programs of expression in distinct compartments of the developing endosperm. The results constitute a preliminary atlas of spatiotemproal patterns of endosperm gene expression in support of future efforts for understanding the underlying mechanisms that control seed yield and quality.

overall_design: The unpollinated kernels (0 DAP), the kernels of 2, 3, 4, 6 DAP and hand dissected endosperms of 8, 10, 12 DAP from the B73 were used to perform high-throughput sequencing using the SOLiD platform

first: Guosheng

last: Li

first: Dongfang

last: Wang

first: Ruolin

last: Yang

first: Kyle

last: Logan

first: Hao

last: Chen

first: Shanshan

last: Zhang

first: Megan

last: Skaggs

first: Alan

last: Lloyd

first: William

middle: J

last: Burnett

first: John

last: Laurie

first: Brenda

last: Hunter

first: Joanne

last: Dannenhoffer

first: Brian

last: Larkins

first: Gary

last: Drews

first: Xiangfeng


title: Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa

city: Raleigh

first: Sermsawat

last: Tunlaya-anukit

state: NC

address: 840 Main campus drive  room2500

department: Department of Forestry and Environmental Resources

institute: North Carolina State University

summary: Laccases were proposed to catalyze the oxidative polymerization of monolignols. We identified 49 laccase gene models in Populus trichocarpa, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all 9 transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an approximately 40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up Graphic Gaussian Model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.

overall_design: Total twelve trees were used. Those include nine individual transgenic trees for overexpressing Ptr-miR397a, as nine biological replicates, and three wild-type trees.

first: Shanfa

last: Lu

first: Quanzi

first: Hairong

last: Wei

first: Mao-Ju

last: Chang

last: Tunlaya-Anukit

first: Hoon

last: Kim

first: Jie

last: Liu

first: Jingyuan

last: Song

first: Ying-Hsuan

last: Sun

first: Lichai

last: Yuan

first: Ting-Feng

last: Yeh

first: Ilona

last: Peszlen

last: Ralph

first: Ronald

last: Sederoff

first: Vincent

middle: L

last: Chiang


title: Next Generation Sequencing of MCF-7 and MDA-MB-435 cells

city: Guangzhou

first: Cuihua

last: Wang

address: 601 Huangpu Ave, Guangzhou

institute: Guangzhou Lab

summary: Human breast cancer cells MDA-MB-435 had higher metastatic potential and other aggressive characteristics than MCF-7 cells.  Next-generation RNA sequencing (RNA-Seq) was used to clear this concern through comparison the transcriptomic expression profiles of these two cells.

overall_design: Total RNA were extracted from MCF-7 and MDA-MB-435S cells, and the polyA+ mRNA was sequenced using Illumina Genome Analyzer IIx. The reads were mapped to RefSeq RNA reference sequences.

first: Xuejuan

last: Gao


title: Circadian behavior is light-reprogrammed by plastic DNA methylation (sequencing)

status: Public on Jan 16 2014

city: Zurich

first: Steven

last: Brown

state: Switzerland

address: Winterthurerstrasse 190

department: Institute of Pharmacology and Toxicology

institute: University of Zurich

summary:
The timing of daily “circadian” behavior can be highly variable among different individuals, and twin studies suggest that about half of this variability is environmentally controlled.  Similar plasticity can be seen in mice exposed to an altered lighting environment – for example, 22-hour days instead of 24-hour ones – which stably alters the genetically determined period of circadian behavior for months.  The mechanisms mediating these environmental influences are unknown.  Here, we show that transient exposure of mice to such lighting stably alters global transcription in the suprachiasmatic nucleus of the hypothalamus (the SCN, the “master clock” tissue determining circadian behavior in mammals). We have also showed that, these changes in transcription are due to change in DNA methylation in the SCN. Indeed, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN.  Importantly, infusion of a methyltransferase inhibitor to the SCN during 22-hour days suppressed period changes.  We also found that these behavioral and DNA methylation changes are reversible upon entrainment to 24-hours days. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity.
Methods: First, 16 mice 5 to 6 weeks old were exposed to DD for 7 days in order to measure their FRP. Then, mice were separated into 2 groups of 8 and entrained to ST and NT cycle. After 4 weeks we recorded wheel-running behavior in constant darkness. After 5 days in DD, we used Clocklab software to predict time of activity onset for each mouse. Brains were isolated at CT4 and sliced with a microtome using a fresh HBSS medium (thickness 250 uM) and the SCN was cut out with the aid of a binocular microscope and stored immediately at –80°C. Total RNA was extracted using the SurePrep™ Nuclear or Cytoplasmic RNA Purification Kit (Fisher Scientific) according to the manufacturer's protocol.  A total of 500 ng of RNA was used for subsequent steps. These steps included DNAase treatment, polyA selection, depletion of ribosomal RNA, overall quality check, and library preparation, and were performed by Fasteris (CH). Libraries were prepared according to standard Illumina protocols.
sequence quality, read mapping and  data analysis: all steps were done using the GeneProf platform. Briefly, the sequencing reads of each library were first quality checked for read length and nucleotide composition, and then submitted for mapping to the mouse genome refrence sequence (Ensemble 58 mouse Genes, NCBIM37 assembly) using the alignment tool TopHat.
Results: Entrainment to non-24 hours light/dark cycle induces a global changes in the SCN transcriptome.

overall_design: SCN mRNA profile  of  mice entrained to normal and short T-cycle using RNAseq technology.

first: Abdelhalim

last: Azzi


title: Transcriptomics analysis of gene expression in normal and METTL3 or WTAP deficient Human HeLa cells

first: Bao-Fa

address: Da-Tun Road

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

summary: RNA was isolated from and METTL3，WTAP deficient Human HeLa cells using the TRIzol (Invitrogen) reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN (RNA integrity number) of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 50 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane.

overall_design: Examination of gene expressive levels in normal and METTL3，WTAP deficient Human HeLa cells

first: Xiaoli

last: Ping

first: Yun-Gui

last: Yang

first: Baofa


title: ZFHX4 and CHD4 ChIP sequencing in glioblastoma tumor initiating cells

city: New York

first: Milan

middle: Girish

last: Chheda

state: NY

address: 408 E. 69th Street

department: Neurology

institute: Memorial Sloan-Kettering Cancer Center

summary: Glioblastomas (GBM) harbor subpopulations of therapy-resistant tumor initiating cells (TICs) that are self-renewing and multipotent. To understand the regulation of the TIC state, we performed an image-based screen for genes regulating GBM TIC maintenance and identified ZFHX4, a 397-kDa transcription factor. ZFHX4 is required to maintain TIC-associated phenotypes in vitro, suggesting that ZFHX4 regulates TIC differentiation, and its suppression increases glioma-free survival in intracranial xenografts. ZFHX4 interacts with CHD4, a core member of the NuRD (nucleosome remodeling and deacetylase) complex. ZFHX4 and CHD4 bind to overlapping sets of genomic loci and control similar gene expression programs. Using expression data derived from GBM patients, we demonstrate ZFHX4 is a master regulator of CHD4-mediated gene expression. These observations define ZFHX4 as a regulatory factor that links the chromatin remodeling NuRD complex and the GBM TIC state.

overall_design: Examination of binding of ZFHX4 and CHD4 across the human genome, using the 0308 tumor initiating cell line. Two replicates for each protein, compared to whole cell extract inputs.


title: Transcriptome Analysis of LincRNA TUNA Knockdown in Mouse Embryonic Stem Cells

status: Public on Feb 03 2014

city: San Diego

first: xiayu

last: huang

address: 3010 Science Park Road

institute: Sanford|Burnham Medical Research Institute

summary: Globel gene expression was analyzed by RNA-seq to study the role of lincRNA TUNA in pluripotent mouse embryonic stem cells.

overall_design: mRNA profiles of CCE mES cells with shRNA-mediated depletion of lincRNA TUNA on days 2, 4, and 6, compared to a control shRNA.

first: Nianwei

last: Lin

first: Tariq

last: Rana


title: METTL3 and WTAP

city: Beijing

last: Sun


title: Distinct roles of Tet1 and Tet2 in mouse embryonic stem cells

city: Heidelberg

first: Lukas

last: Chavez

address: Im Neuenheimer Feld 280

country: Germany

institute: German Cancer Research Center (DKFZ)


title: Identification of zygotically transcribed genes in zebrafish by 4-thio-UTP metabolic labeling

city: Dresden

first: Patricia

last: Heyn

address: Pfotenhauerstrasse 108

institute: Max Planck Institute of Molecular Cell Biology and Genetics

summary: The first embryonic cell divisions rely on maternally stored mRNA and proteins. The zygotic genome is initially transcriptionally silenced and activated later in a process called zygotic genome activation (ZGA). ZGA in any species is still a poorly understood process; the timing of transcription onset is controversial and the identity of the first transcribed genes unclear. Zebrafish, Danio rerio, is a rapidly developing vertebrate model, which is accessible to experimentation and global studies before, during and after ZGA.

overall_design: To accurately determine the onset of ZGA and to identify the first transcripts in zebrafish, we developed a metabolic labeling method, utilizing the ribonucleotide analog 4-thio-UTP, which allows efficient and specific affinity purification of newly transcribed RNA. Using deep sequencing, we characterized the onset of transcription in zebrafish embryos at 128-, 256-, and 512-cell stages. We identified 592 nuclear-encoded zygotically transcribed genes, comprising 670 transcript isoforms. Mitochondrial genomes were highly transcribed at all time points. Further, bioinformatic analysis revealed an enrichment of transcription factors and miRNAs among the newly transcribed genes, suggesting mechanistic roles for the early genes that are required to activate subsequent gene expression programs in development. Interestingly, analysis of gene-architecture revealed that zygotically transcribed genes are often intronless and short, reducing transcription and processing time of the transcript. The newly generated dataset enabled us to compare zygotically transcribed genes over a broad phylogenetic distance with fly and mouse early zygotic genes. This analysis revealed that short gene length is a common characteristic for early zygotically expressed genes. However, we detected a poor level of overlap for shared orthologs.

first: Karla

last: Neugebauer


title: Genome-wide map of RNA polymerase II, NELF, and DSIF in HeLa cells

status: Public on Feb 01 2014

city: Iowa City

first: David

last: Price

state: IA

address: 500 Newton Road

department: Biochemistry

institute: University of Iowa

summary: Most metazoan promoters have RNA polymerase II (Pol II) paused slightly downstream of the transcription start site by NELF and DSIF. This pausing keeps these promoters available for rapid induction by P-TEFb, whose activity causes NELF to dissociate and Pol II and DSIF to elongate on the gene. ChIP-Seq data was generated for Pol II, NELF, and DSIF in HeLa cells and used to look at pausing downstream of unannotated promoters.

overall_design: 3x ChIP-Seq (Pol II, NELF, DSIF) in HeLa cells

first: Kyle

middle: A

last: Nilson

middle: H


title: Distinct roles of Tet1 and Tet2 in mouse embryonic stem cells (CMS-Seq)

summary: The TET proteins TET1, TET2 and TET3 constitute a new family of dioxygenases that utilize molecular oxygen and the cofactors Fe(II) and 2-oxoglutarate to convert 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC) and further oxidation products in DNA1-5. Here we show that Tet1 and Tet2 have distinct roles in regulating 5hmC deposition and gene expression in mouse embryonic stem cells (mESC). Tet1 depletion in mESC primarily diminishes 5hmC levels at transcription start sites (TSS), whereas Tet2 depletion is mostly associated with decreased 5hmC in gene bodies relative to TSS. 5hmC is enriched at exon start and end sites, especially in exons that are highly expressed, and is significantly decreased upon Tet2 knockdown at the boundaries of high-expressed exons that are selectively regulated by Tet2. In differentiating murine B cells, Tet2 deficiency is associated with selective exon exclusion in the gene encoding the transmembrane phosphatase CD45. Tet2 depletion is associated with increased 5hmC and decreased 5mC at promoters/ TSS regions, possibly because of the redundant activity of Tet1. Together, these data indicate a complex interplay between Tet1 and Tet2 in mESC, and show that loss-of-function of a single TET protein does not necessarily lead to loss of 5hmC and a corresponding gain of 5mC, as generally assumed. The relation between Tet2 loss-of-function and selective changes in exon expression could potentially explain the frequent occurrence of both TET2 loss-of-function mutations and mutations in proteins involved in pre-mRNA splicing in myeloid malignancies in humans.

overall_design: Gene and exon expression analysis in mESC, Tet1 knockdown mESC, and Tet2 knockdown mESC by RNA-sequencing. Mapping of 5-hydroxymethylcytosine in mESC, Tet1 knockdown mESC, and Tet2 knockdown mESC by anti-CMS-seq. Mapping of methylcytosine in mESC, and Tet2 kd mESC by MeDIP-seq.

first: Yun

last: Huang

first: Anjana

last: Rao


title: Distinct roles of Tet1 and Tet2 in mouse embryonic stem cells (RNA-Seq)


title: Otx2 ChIP-seq in the adult mouse Retinal Pigmented Epithelium (RPE) and neural retina

status: Public on Jan 15 2014

city: Nice

first: Alexander

last: Samuel

address: Parc Valrose

department: Institute of Biology Valrose

institute: CNRS, INSERM, Université de Nice Sophia-Antipolis

summary: Otx2 has been shown to be non cell autonomously required for photoreceptor cell survival in the adult mouse RPE. This study aims to identify Otx2 DNA binding profile in both RPE and neural retina to i) identify direct targets of Otx2 in the RPE ii) compare Otx2 binding profile in neural retina and RPE to unveil hidden functions in the neural retina.

overall_design: WT and GFP antibodies were used to perform two independent ChIP-seq experiments using Illumina GAIIx.

first: Thomas

last: Lamonerie

first: Michael

last: Housset


title: Obesity, rather than diet, drives epigenomic alterations in colonic epithelium resembling cancer progression

city: RTP

first: ruifang

last: li

address: 111 T.W. Alexander Drive

institute: NIEHS

summary: High-fat diet and obesity are high risk factors for colorectal cancer. The underlying mechanism is still unclear. Environmental factors alter the epigenome to affect gene expression thus the phenotype. In response to external stimuli, the cis-regulatory regions, especially enhancer loci, are key elements for regulating selective gene expression. We thus explored the effects of high-fat diet and the accompanying obesity on gene expression and the enhancer landscape in colon epithelium. High-fat diet exposed binding sites of transcription factors downstream of signaling pathways important in the initiation and progression of colon cancer. Meantime, colon specific enhancers were lost rendering the cells potential for dedifferentiation. The alteration at enhancer regions drives a specific transcription program promoting colon cancer progression. The comprehensive interrogation of enhancer changes by high-fat diet in colon epithelium provides a number of insights into the underlying biology of high-fat diet and obesity in increasing colon cancer risk, and provides potential therapeutic targets to treat obese colon cancer patients.

overall_design:
ChIP sequencing of active enhancer mark h3k27ac in colon epithelium from wild type mice and NAG-1 transgenic mice treated with either low-fat diet or high-fat diet.

The gene expression component of the study is included in GSE46843.

first: Ruifang

last: Li

first: Paul

last: Wade

first: Sara

last: Grimm


title: Transcriptome analysis of yeast methyltransferase mutants set5∆, set1∆ and set5∆set1∆

city: Stanford

first: Gloria

last: Mas Martin

address: 371 Serra Mall

institute: Stanford University

summary:
Purpose: Understand the synergistic relationship between the methyltransferases Set1 and Set5 in the regulation of gene expression.
Methods: Total mRNA was obtained from two independent biological replicates each of wildtype (WT), set1∆, set5∆, SET5 Y402A and set1∆set5∆  S. cerevisiae strains. Libraries were generated and sequenced using an Illumina HiSeq2000 platform. The sequence reads that passed quality filters were mapped using TopHat and expression levels were quantified using Cufflinks.
Results: We generated FPKM expression values for each transcript and identified the differentially expressed genes using an FDR-adjusted p-value of 0.05. Subsequent data analysis was restricted to genes with fold-change greater than 1.7 relative to WT. Our results show that Set1 and Set5 have roles primarily in transcription repression. Moreover, lack of both Set1 and Set5 results in a synergistic exhacerbation of the transcriptional derepression observed in the single mutants. Further analysis revealed a specific enrichment of the Set5/Set1-repressed genes near repetitive DNA sequences of the genome.
Conclusions: Our study uncovers an unexpected synergistic role of Set1 and Set5 in transcription repression of telomeric regions and Ty retrotransposons.

overall_design: mRNA profiles of wildtype (WT), set1∆, set5∆, SET5 Y402A and set1∆set5∆ were generated by sequencing using an Illumina HiSeq2000 platform. Two biological replicates of each strain were used.

last: Martin

first: Devin

last: King

first: Pablo

middle: E

last: Garcia-Nieto


title: Genome-wide identification of Hfq-regulated small RNAs in the bacterial pathogen Erwinia amylovora

city: East Lansing

first: Quan

last: Zeng

state: MI

address: 578 Wilson Rd. CIPS103

department: Plant, Soil, and Microbial Sciences

institute: Michigan State University

summary: RNA-seq analysis was performed to examine the sRNA transcripts in wild type E. amylovora 1189 and in the deletion mutant of hfq, at 6 and 12 hours of culture in hrp-inducing minimal medium.

overall_design: Total RNA was isolated from wild type Ea1189, and the hfq mutant of Ea1189, at 6 and 12 hours post inoculation in hrp-inducing minimal medium.  RNA transcripts that are between 50-350 nucleotides were analyzed by RNA-seq.  Intergenic transcripts whose expression was greatly reduced in the hfq mutant than the wild type at both 6 and 12 hours post inoculation in hrp-inducing minimal medium were selected as potential small RNAs.

first: Kevin

middle: M

last: Carr

first: George

last: Sundin


title: ChIP-seq of MDA-MB-134 cell line

city: Pittsburgh

first: Amir

last: Bahreini

state: PA

address: 204 Craft Ave A430

department: Human Genetics

institute: University of Pittsburgh

summary: We performed ChIP seq experiment in MDA-MB-134 cell line in order to map the estrogen receptor alpha (ER) binding sites following the estrogen treatment in an ILC model. We have characterized the genome wide recruit of ER and scaned the binding sites for the presence of cofactor motifs. The binding peaks were also correlated to E2 regulated genes in this ILC model.

overall_design: Four samples were subjected to high throughput sequencing: E-ER (estrogen treated followed by ER IP), E-IgG (estrogen treated followed by IgG), V-ER (EtOH treated followed by ER IP) and Input (MCF7 genomic DNA)

first: Matthew

last: Sikora


title: Abeta levels in APP Tg mice with mixed genetic backgrounds

city: Suita, Osaka

first: Noriyuki

last: Hayashi

address: G9, 2-2, Yamadaoka,

department: Complementary and Alternative Medicine

country: Japan

institute: Osaka University Graduate School of Medicine

summary: We combined a mouse model of Abeta accumulation and transcriptomics, and identified Klc1 as an Abeta accumulation modifier in vivo.

overall_design: For this transcriptomics analysis, we used 12 arrays (one mouse mRNA sample per array) for inbred mice analyses and 28 arrays for the APP Tg mice of mixed genetic backgrounds.  First, 13309 probes whose signals were reliably detectable in all 40 arrays were selected from 25967 probes on the Illumina mouse Ref-8 Expression BeadChip.  Second, to select the probes whose expression levels were affected by the DBA genetic background, we compared the expression levels of the 13309 probes in DBA, B6 and SJL inbred (non-Tg) mice (unpaired t-test).　Using inbred mice means that any change in gene expression is based on their genetic background, not the secondary effects of Abeta accumulation. To minimize the chance of false positives, we applied strict criteria in this selection: the fold change was equal to or more than 1.5 and the false discovery rate (FDR) was set to 0.001. In total 54 probes were identified, with the signals of 47 probes being lower and 7 probes being higher in DBA mice than those in either B6 or SJL.   In the final step, we examined the correlation between the expression levels of these 54 probes and Abeta40 levels in the GuHCl fraction in APP Tg mice. Using strict selection criteria (Pearson’s product-moment correlation FDR=0.001), we identified a total of four probes which correlated with Abeta levels. Two of these probes were higher in DBA and negatively correlated with the levels of Abeta accumulation. The other two probes were lower in DBA and positively correlated with the levels of Abeta accumulatio. Notably, the two probes (probe ID 4050133 and 6130468) that positively correlated with Abeta accumulation both detected the same transcript: kinesin light chain 1 (Klc1; also known as Kns2).

first: Takashi

last: Morihara

first: Mikiko

last: Yokokoji


title: HoxC11 ChIP-seq of LY2 Breast Cancer Cell Line

status: Public on Jan 14 2014

city: Dublin

first: Leonie

last: Young

address: York Street

department: Surgery

country: Ireland

institute: Royal College of Surgeons, Ireland

summary: SRC-1 (NCOA1) is a steroid receptor coactivator that has been associated with various aspects of the progression of breast cancer disease such as tamoxifen resistance, metastasis, cell proliferation and invasiveness.  In a tamoxifen resistant breast cancer cell line (LY2), SRC-1 has been found to interact with the developmental transcription factor HoxC11. ChIP-sequencing of HoxC11 in LY2 cells shows where the transcription factor binds throughout the genome.

overall_design: LY2 cells were treated with either tamoxifen or vehicle and immunoprecipitated with anti-Hoxc11

first: Yuan

last: Hao

first: Ailis

last: Fagan


title: Enhancer landscape of mouse developing midbrain at 11.5 dpc embryos

city: Yokohama

first: Takaho

middle: A.

last: Endo

state: Kanagawa

address: 1-7-22 Suehiro, Tsurumi

department: IMS

institute: RIKEN

summary: We report the distribution of histone H3K4me1 and H3K27ac within the genome of mouse embryonic midbrain. We prepared the chromatin from 11 dpc embryonic  midbrain and made chromatin precipitation with antibody against H3K4me1 and H3K27ac (rabbit polyclonal antibody). High-throughput sequencing applied for the ChIP analysis revealed the differential distribution of modified histone within developing midbrain.

overall_design: ChIP analysis of mouse 11 dpc embryonic midbrain and forelimb buds against anti-H3K4me1 and H3K27ac antibody.

last: Kondo

first: Haruhiko

last: Koseki


title: RING1B binding in the 11 dpc embryonic tissues

summary: We report the distribution of RING1B (one of Polycomb group factors) within the genome of mouse embryonic tissues. We prepared the chromatin from 11 dpc embryonic forebrain, midbrain, and forelimb buds and made chromatin precipitation with antibody against RING1B (mouse monoclonal antibody). High-throughput sequencing applied for the ChIP analysis revealed the differential distribution of RING1B protein among embryonic tissues.

overall_design: ChIP analysis of mouse 11 dpc embryonic forebrain, midbrain and forelimb buds against anti-RING1B antibody.


title: MITF ChIP-seq in primary melanocyte and melanoma as a function of oncogenic BRAF

first: Paul

last: Khavari

state: CA

address: 269 Campus Drive

summary: Thousands of enhancers are characterized in the human genome, yet few have been shown important in cancer. Inhibiting oncokinases, such as EGFR, ALK, HER2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by upregulation of the HGF receptor, MET. The mechanisms mediating such genomic responses to targeted therapy are unknown. Here, we identify lineage-specific MET enhancers for multiple common tumor types, including a melanoma lineage-specific MET enhancer that displays inducible chromatin looping and MET gene induction upon BRAF inhibition. Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function. Targeted genomic deletion (<7bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation. Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of tumor lineage-enriched transcription factors mediating innate resistance to oncokinase therapy.

overall_design: MITF ChIP-seq was performed in primary human melanocytes with overexpression of BRAFV600E or a lentiviral control (RFP), and in COLO829 melanoma cells treated with DMSO, or PLX4032

first: Dan

last: Webster


title: Role of MITF in melanoma

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

overall_design: Refer to individual Series


title: Binding motif analysis of METTL3，WTAP in METTL3，WTAP overexpressed Human 293T cells

first: Bao-Fa

last: Sun

summary: label the cells overexpressed Myc tagged METTL3 and Flag tagged WTAP with 4-SU, the RNA bound by METT3,WTAP can be got by Myc or Flag IP followed by RNA isolation by using the TRIzol (Invitrogen) reagent by following the company manual.the RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 50 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane.

overall_design: discovery of the binding motif of METTL3，WTAP in METTL3，WTAP overexpressed Human 293T cells

first: Xiaoli

last: Ping

first: Yun-Gui

last: Yang

first: Baofa


title: Brd4 and JMJD6-associated Anti-pause Enhancers in Regulation of Transcriptional Pause Release

first: Qi

last: Ma

address: 9500 Gilman Dr,

institute: UCSD

summary: Distal enhancers characterized by H3K4me1 mark play critical roles in developmental and transcriptional programs. However, potential roles of specific distal regulatory elements in regulating RNA Polymerase II (Pol II) promoter-proximal pause release remain poorly investigated. Here we report that a unique cohort of jumonji C domain-containing protein 6 (JMJD6) and bromodomain-containing protein 4 (Brd4) co-bound distal enhancers, termed anti-pause enhancers (A-PEs), regulate promoter-proximal pause release of a large subset of transcription units via long-range interactions. Brd4-dependent JMJD6 recruitment on A-PEs mediates erasure of H4R3me2(s), which is directly read by 7SK snRNA, and decapping/demethylation of 7SK snRNA, ensuring the dismissal of the 7SKsnRNA/HEXIM inhibitory complex. The interactions of both JMJD6 and Brd4 with the P-TEFb complex permit its activation and pause release of regulated coding genes. The functions of JMJD6/ Brd4-associated dual histone and RNA demethylase activity on anti-pause enhancers have intriguing implications for these proteins in development, homeostasis and disease.

overall_design:
All Gro-seq(s) were designed to reveal the transcriptional targets of JMJD6 and Brd4, and assess the role of JMJD6 and Brd4 in Pol II promoter-proximal pause release.
All ChIP-seq(s) were designed to understand the unique features, associated molecular mechanisms and functions of the anti-pause enhancers (A-PEs) discovered in the current study.

first: Wen

last: Liu


title: Expression data from rice leaves treated or not with fungal elicitors and high-throughput pyrosequencing of endogenous small RNAs from Oryza sativa

city: Bellaterra (Cerdanyola del Vallés)

first: Sonia

last: Campo

state: Barcelona

address: Parc de Recerca UAB Edifici CRAG, Campus UAB

country: Spain

institute: Center for Research in Agricultural Genomics (CRAG)

summary:
High-throughput sequencing of small RNAs from rice was used to identify distinct miRNAs that are responsive to elicitors from the fungal pathogen Magnaporthe oryzae.

[Expression profiling by array] We used microarrays to determine the expression behaviour of target genes for elicitor-regulated miRNAs.

[High throughput sequencing] High-throughput sequencing of rice small RNAs was performed in two different tissues, leaves and roots, and two different time point of elicitor treatment, 30' and 2h
Amplicons were prepared by 5´and 3´adaptor ligation in which the 5'-adaptor contained a 'barcode' consisting of a 4-nucleotide identifier sequence for each sample. The libraries containing unique barcodes were combined and subjected to pyrosequencing (454 Life SciencesTM, Roche)

platform_organism:
Oryza sativa
Oryza sativa Japonica Group

overall_design:
[Expression profiling by array] Leaves from rice plants were harvested at two time points after the onset of treatment (30' and 2h) with elicitors of Magnaporthe oryzae 18.1 and used for RNA extraction and hybridization on Affymetrix microarrays. Mock inoculations were performed with sterile water for control experiments. Three biological replicates were analyzed. Each sample represented a pool of approximately 150 rice plants.

[High throughput sequencing] 8 samples examined: leaves and roots, treated or not with elicitors at two different time points, 30' and 2h (2x2x2)

first: Christelle

last: Siré

first: Ana

middle: B

last: Moreno

first: Livia

last: Donaire

first: Matthias

last: Zytnicki

first: Cedric

last: Notredame

first: César

last: Llave

first: Blanca

last: San Segundo


title: Interplay of choline metabolites and genes in patient-derived breast cancer xenografts

city: Oslo

first: Einar

middle: Andreas

last: Rødland

address: Montebello

department: Institute for Cancer Research

country: Norway

institute: Oslo University Hospital HF - Radiumhospitalet

summary: Dysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism.   Methods: Tumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analysed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analysed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson’s correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups.   Results: Most of the xenograft models were classified as basal-like (N=19) or luminal B (N=7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5).  These characteristics were similar to those found in human tumor samples.  Discussion: The higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathway-encoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo.

overall_design: Gene expression was measured in 30 human breast cancer xenografts, one sample from each model

first: Maria

middle: T

last: Grinde

first: Nirma

last: Skrbo

first: Siver

last: Moestue

first: Eldrid

last: Borgan

first: Alexandr

last: Kristian

first: Beathe

last: Sitter

first: Tone

last: Bathen

first: Anne-Lise

last: Børresen-Dale

first: Gunhild

last: Mælandsmo

first: Olav

last: Engebråten

first: Therese

last: Sørlie

first: Elisabetta

last: Marangoni

first: Ingrid

middle: S

last: Gribbestad


title: 4C-seq indicate interactive sites with the sequence close from Meis2 promoter

first: Takaho

last: Endo

summary: We report the distribution of interactive sites with the sequence close from Meis2 promoter within the genome of mouse embryonic forebrain. We prepared the chromatin from 11 dpc embryonic forebrain and made 3C (chromosomal conformation capture) library. High-throughput sequencing applied for the 3C analysis revealed the distribution of modified interactive sites within developing forebrain.

overall_design:
4C-seq analysis of mouse 11 dpc embryonic forebrain with the sequence close from Meis2 promoter.
Forebrain isolated and disected from 11 dpc embryos are fixed by 1% formaldehyde. After conventional 3C reaction, 3C library for highthroughput sequence is prepared by combination of adaptor ligation and nesting PCR reactions.

first: Takashi

last: Kondo

first: Haruhiko

last: Koseki


title: OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness

status: Public on Jan 13 2014

city: Saclay

first: Jerome

middle: Eric

last: Roger

state: Please select

address: 151 Route de la Rotonde, Campus CEA Saclay - Bat 151

department: CERTO

institute: CNRS-Universite Paris Sud

summary: Leber congenital amaurosis (LCA) includes congenital or early-onset blinding diseases, characterized by vision loss together with nystagmus and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA. While most LCA is recessive, mutations in the homeodomain transcription factor gene CRX lead to autosomal dominant LCA. The mechanism of CRX-LCA is not understood. Here, we report a new spontaneous mouse mutant carrying a frameshift mutation in Crx (CrxRip). We show that, unlike Crx-/- mouse retina, the dominant Crx c.763del1 mutation in CrxRip results in congenital blindness with complete loss of ERG, yet the photoreceptors do not degenerate. Dominant CRX frameshift mutations associated with LCA mimic the CrxRip phenotype that can be rescued by Crx. RNA-Seq profiling reveals progressive and complete loss of rod differentiation factor Nrl in CrxRip, while residual Nrl remains in Crx-/- retina. Moreover, Nrl partially restores the rod phenotype in CrxRip/+ mice. We show that the binding of Otx2 to Nrl promoter is obliterated in CrxRip mutant, and ectopic Otx2 can rescue the rod phenotype. Therefore, Otx2 is required to maintain Nrl expression in developing rods to consolidate rod fate. Our studies provide the mechanism of congenital blindness caused by dominant CRX mutations and should assist in therapeutic design.

overall_design: Retinal samples were harvested from WT, CrxRip/+, CrxRip/Rip, Crx-/- and Nrl-/- retina at postnatal days 2 and 21 for whole transcriptome sequencing (RNAseq).  Each sample included 2 independent frozen retina and experiments were performed in duplicates. RNA-seq transcriptome libraries were constructed from 1 μg of total RNA.

first: Avinash

last: Hiriyanna

first: Norimoto

last: Gotoh

first: Hong

last: Hao

first: Debbie

last: Cheng

first: Rinki

last: Ratnapriya

first: Marie-Audrey

middle: I

last: Kautzmann

first: Bo

last: Chang

first: Anand

last: Swaroop


title: Extracellular Vesicles in Luminal Fluid of the Ovine Uterus

status: Public on Jan 11 2014

city: Columbia

first: Thomas

last: Spencer

state: MO

address: 920 East Campus Drive

department: Animal Sciences

institute: University of Missouri

summary: Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Extracellular vesicle RNA was pooled (n=4 per status) and analyzed for small RNAs by sequencing on the Ion Torrent PGM platform and analysis with CLC Genomics Workbench small RNA workflow based on the miRBase (Release 19) Bos taurus  database.

overall_design: Small RNA analysis of day 14 uterine luminal fluid extracellular vesicles isolated from pregnant and cyclic ewes.

first: Gregory

middle: W

last: Burns

first: Kelsey

last: Brooks

first: Mark

middle: R

last: Wildung

first: Raphatphorn

last: Navakanitworakul

first: Lane

last: Christenson


title: Transcriptome sequencing to systematically detect trans-splicing in human embryonic stem cells

city: Taipei

first: Chan-Shuo

last: Wu

address: 128 Academia Road, Section 2, Nankang

department: Genomics Research Center

country: Taiwan

institute: Academia Sinica

summary: Trans-splicing occurs post-transcriptionally and generates transcripts that are orderly inconsistent with their corresponding DNA templates. Until recently only exceedingly rare trans-splicing events have been experimentally characterized in the mammalian transcriptomes. Although hundreds to thousands of trans-spliced RNA candidates have been nominated by bioinformatics- or NGS (next-generation sequencing)-based approaches, these candidates unavoidably suffered from potential false positives arising from genetic rearrangement events or in vitro artifacts. Here we develop a pipeline (TSscan) based on NGS transcriptome data to identify trans-splicing in human embryonic stem cells (ESCs). TSscan integrates RNA sequencing data derived from different NGS platforms (i.e., Roche 454, SOLiD, and Illumina) and different human ESC lines (i.e., H1 and H9) as well as several in silico filters to minimize these two types of potential false positives. Our result shows that a tremendous amount of apparent experimental artifacts are indeed present in NGS data, which may be the most major false positives of trans-splicing detection. TSscan totally identified 10 trans-spliced RNA candidates in human ESCs, four of which are experimentally validated to be true. Further experiments reveal that these four events represent differential expression during the transition of pluripotent status to differentiate statuses. Especially, we observe that one event (the trans-spliced isoform of NCRMS), which is also a large intergenic non-coding RNA, tends to be specifically transcribed in ESCs and induced pluripotent stem cells and can conspicuously affect the pluripotency maintenance of ESCs. As far as we know, TSscan is the first pipeline for systematic identification of trans-splicing that utilizes NGS data in the human transcriptome, opening up an important class of post-transcriptional events for comprehensive characterization.

overall_design: human embryonic stem cell H9

first: Trees-Juen

last: Chuang


title: Transcriptome In Vivo Analysis (TIVA) of spatially defined single cells in intact live mouse and human brain tissue

status: Public on Jan 12 2014

city: Philadelphia

first: Junhyong

last: Kim

address: 433 S University Avenue

department: Biology

institute: University of Pennsylvania

summary: Transcriptome profiling is an indispensable tool in advancing the understanding of single cell biology, but depends upon methods capable of isolating mRNA at the spatial resolution of a single cell. Current capture methods lack sufficient spatial resolution to isolate mRNA from individual in vivo resident cells without damaging adjacent tissue. Because of this limitation, it has been difficult to assess the influence of the microenvironment on the transcriptome of individual neurons. Here, we engineered a Transcriptome In Vivo Analysis (TIVA)-tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA-tag in combination with RNA-seq to analyze transcriptome variance among single dispersed cells and in vivo resident mouse and human neurons, we show that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology provides the first noninvasive approach for capturing mRNA from single cells in their natural microenvironment.

overall_design: Samples represent cortex and hippocampus neuron cells collected by pipette and TIVA captured.

first: Ditte

last: Lovatt

first: Brittani

middle: K

last: Ruble

first: Jaehee

last: Lee

first: Hannah

last: Dueck

first: Julia

last: Richards

first: Tae

first: Stephen

last: Fisher

first: Chantal

last: Francis

first: Jennifer

last: Spaethling

first: John

last: Wolf

first: M

last: Grady

first: Alexandra

middle: V

last: Ulyanova

first: Julianne

middle: C

last: Greipenbrug

first: Peter

last: Buckley

first: Jai-Yoon

last: Sul

first: Ivan

last: Dmochowski

first: Jim

last: Eberwine


title: Identification of a novel Bromodomain / Casein Kinase II / TAF-containing complex as a regulator of mitotic condensin function

status: Public on Jan 10 2014

city: New York

first: Michael

middle: Christopher

last: Keogh

state: New York

address: 1300 Morris Park Avenue

department: Department of Cell Biology

institute: Albert Einstein College of Medicine

summary: ABSTRACT: Condensin is a central regulator of mitotic genome structure, with mutants showing poorly condensed chromosomes and profound segregation defects. Here we identify the fission yeast NCT complex, comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), Casein Kinase II (CKII) and several TAFs, as a novel regulator of condensin function (where NCT mutants restore the formation of segregation-competent chromosomes in cells containing defective condensin). Synchronous ChIP-seq shows that NCT and condensin bind similar genomic regions, but only briefly co-localize during the periods of chromosome condensation and decondensation. These results are consistent with a model where NCT targets CKII to chromatin in a cell cycle-directed manner to modulate the activity of condensin during chromosome condensation and decondensation.

overall_design: DATA:  Study includes ChIP-seq of fission yeast H3-K4Me3, H3-K36Me3, TBP, Taf7, Nrc1, Cka1 from aynchronous cells; Nrc1 and Cut3 (representing condensin) from four synchronized cell-cycle stages estimated as G2/M, Metaphase, Anaphase and G1/S.

first: Michael-Christopher

first: Hyun-Soo

last: Kim

first: Rituparna

last: Mukhopadhyay


title: RNASeq of Developing Arabidopsis Seeds Producing Hydroxy-Fatty Acids

first: Kevin

last: Carr

address: Plant Biology Lab, 612 Wilson Rd., Rm. S-20 A

department: Research Technology Support Facility

summary: Arabidopsis seeds expressing the castor fatty acid hydroxylase accumulate hydroxylated fatty acids up to 17% of total fatty acids in seed triacylglycerols, however total seed oil is also reduced up to 50%. Investigations into the cause of the reduced oil phenotype through in vivo [14C]acteate and [3H]2O metabolic labeling of developing seeds surprisingly revealed that the rate of de novo fatty acid synthesis within the transgenic seeds was approximately half that of control seeds. Addition of castor phospholipid:diacylglycerol acyltransferase (PDAT) increased hydroxylated fatty acid content of the seed oil, increased the rate of fatty acid synthesis, and mostly restored seed oil levels. RNAseq analysis indicated no changes in expression of fatty acid synthesis genes in hydroxylase-expressing plants.

overall_design: Transcript profiles of Arabidopsis developing seeds of three lines, at three stages of development were generated by deep sequencing, in triplicate, using Illumina.

first: Philip

middle: D

last: Bates

last: Ohlrogge


title: RNA-seq of poly(A)-/ribo- or poly(A)+ RNAs from mouse and rhesus ES cells

city: Shanghai

first: Li

last: Yang

address: 131 Dong-An Road

department: Institutes of Biological Sciences

institute: Fudan University

summary: We have used RNA-seq to examine circular RNAs from RNase R treated poly(A)-/ribo- RNAs in human embryonic stem cells

sample_organism:
Macaca mulatta
Mus musculus

platform_organism:
Macaca mulatta
Mus musculus

overall_design: to identify novel lncRNAs from different species

first: Xiao-Ou

last: Zhang

first: Qing-Fei

last: Yin

first: Yang

first: Ling-Ling

last: Chen


title: Technical Variations in Low-Input RNA-seq Methodologies

city: la jolla

first: vipul

last: bhargava

state: California

address: 9500 gilman drive

institute: ucsd

summary: Transcriptomics data obtained from limiting amounts of mRNA is often noisy, providing primarily qualitative changes in transcript expressions. So far, technical variations arising out of the library preparation protocols have not been adequately characterized at reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, stochasticity in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations, which were sequencing depth independent, ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.

overall_design: Sequencing libraries were prepared from serial dilutions of mRNA, ranging from 1 ng to 25 pg, using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq. The mRNA was derived from an in vitro model of lineage segregation achieved by modulating TGF beta signaling pathway in differentiating mouse embryonic stem cells.

first: Vipul

last: Bhargava

first: Shankar

last: Subramaniam