title: Transcription factor occupancy is linked to DNA methylation turnover at active regulatory regions [Bisulfite-Seq]

first: Dirk

last: Schuebeler

overall_design: CTCF ChIP bisulfite sequencing in mouse embryonic stem cells and whole genome shotgun bisulfite sequencing of RESTko mouse embryonic stem (ES) cells

first: Robert

last: Ivanek

first: Angelika

last: Feldmann

first: Rabih

last: Murr

first: Dimos

last: Gaidatzis

first: Lukas

last: Burger

last: Schübeler


title: dMyc_Kc167_cells_ChIP-seq

status: Public on Dec 21 2013

city: Toronto

first: DCC

last: modENCODE

state: Ontario

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

country: Canada

institute: Ontario Institute for Cancer Research

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
modENCODE_submission_5006
This submission comes from a modENCODE project of Kevin White. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: The White Lab is aiming to map the association of all the Transcription Factors (TF) on the genome of Drosophila melanogaster. One technique that we use for this purpose is chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) utilizing an Illumina next generation sequencing platform. The data generated by ChIP-seq experiments consist basically of a plot of signal intensity across the genome. The highest signals correspond to positions in the genome occupied by the tested TF.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Cell Line: Kc167; Tissue: embryo-derived cell-line; Developmental Stage: late embryonic stage; Genotype: se/e; Sex: Female;  EXPERIMENTAL FACTORS: Antibody MycN (target is fly genes:dm); Cell Line Kc167

first: Kevin

last: White

first: Lijia

last: Ma

first: Matthew

last: Slattery


title: HP1_8-16-seq

summary:
modENCODE_submission_4091
This submission comes from a modENCODE project of Kevin White. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: The White Lab is aiming to map the association of many of the non-histone chromosomal proteins on the genome of Drosophila melanogaster. One technique that we use for this purpose is chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) utilizing an Illumina next generation sequencing platform. The data generated by ChIP-seq experiments consist basically of a plot of signal intensity across the genome. The highest signals correspond to positions in the genome occupied by the tested chromatin-binding protein.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Y cn bw sp; Developmental Stage: Embryo 8-16h; Genotype: y[1] oc[R3.2]; Gr22b[1] Gr22d[1] cn[1] CG33964[R4.2] bw[1] sp[1]; LysC[1] lab[R4.2] MstProx[1] GstD5[1] Rh6[1];  EXPERIMENTAL FACTORS: Developmental Stage Embryo 8-16h; Strain Y cn bw sp; Antibody HP1 antibody (ab24726) (target is HP1)


title: Zrf1 is required to establish and maintain neural progenitor identity (ChIP-Seq)

city: Barcelona

first: Luciano

last: Di Croce

address: Dr. Aiguader, 88

country: Spain

institute: Centre for Genomic Regulation

summary: We report ChIP-seq data for Zrf1 and Ring1B occupancy in NPC

overall_design: Examination and comparison of DNA binding profile of Zrf1 and Ring1B in NPC

first: Luigi

last: Aloia

first: Luca

last: Cozzuto


title: Su(H)_Kc_cells_ChIP-seq

summary:
modENCODE_submission_5018
This submission comes from a modENCODE project of Kevin White. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: The White Lab is aiming to map the association of all the Transcription Factors (TF) on the genome of Drosophila melanogaster. One technique that we use for this purpose is chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) utilizing an Illumina next generation sequencing platform. The data generated by ChIP-seq experiments consist basically of a plot of signal intensity across the genome. The highest signals correspond to positions in the genome occupied by the tested TF.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Cell Line: Kc167; Tissue: embryo-derived cell-line; Developmental Stage: late embryonic stage; Genotype: se/e; Sex: Female;  EXPERIMENTAL FACTORS: Antibody Su(H) (target is fly genes:Su(H)); Cell Line Kc167


title: Transcriptome analysis of Mouse E14-TG2a.IV ES cells

city: oxford

first: Jim

last: Hughes

address: Weatherall Institute Of Molecular Me

department: MHU

country: United Kingdom

institute: University of Oxford

type: Expression profiling by high throughput sequencing

summary: Analysis of gene expression in Mouse E14-TG2a.IV strain ES cells

overall_design: Single end RNA-seq analysis of PolyA selected RNA from E14-TG2a.IV ES cells

first: Magnus

last: Lynch

first: Doug

last: Higgs


title: Zrf1 is required to establish and maintain neural progenitor identity

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

overall_design: Refer to individual Series


title: H_Kc_cells_ChIP-seq

summary:
modENCODE_submission_4983
This submission comes from a modENCODE project of Kevin White. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: The White Lab is aiming to map the association of all the Transcription Factors (TF) on the genome of Drosophila melanogaster. One technique that we use for this purpose is chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) utilizing an Illumina next generation sequencing platform. The data generated by ChIP-seq experiments consist basically of a plot of signal intensity across the genome. The highest signals correspond to positions in the genome occupied by the tested TF.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Cell Line: Kc167; Tissue: embryo-derived cell-line; Developmental Stage: late embryonic stage; Genotype: se/e; Sex: Female;  EXPERIMENTAL FACTORS: Antibody KW3-h-D1 (target is Hairy); Cell Line Kc167


title: Multiplexed Chromatin Conformation Capture in Mouse Erythroid cells using Capture-C

summary: Multiplexed Chromatin Conformation Capture in Mouse Erythroid cells , from hundreds of targeted loci,  using agilent oligo capture technology and high throughput sequencing.

overall_design: Two erythroid Ter119+ cell replicates and a mouse ES cell control


title: INTERACTIONS BETWEEN CYANOBACTERIA AND HETEROTROPHS UNDER DIFFERENT TROPHIC CONDITIONS

status: Public on Dec 20 2013

city: Richland

first: Lye Meng

last: Markillie

state: WA

address: 902 Battelle Blvd

department: Biosystems Dynamics & Simulation

country: USA

institute: Environmental Molecular Sciences Laboratory

summary: Application of genome-scale 'omics approaches to dissect subcellular pathways and regulatory networks governing interspecies interactions is dependent, at least initially, on the availability of model systems with well-annotated genomes and tractable genetics.

sample_organism:
Picosynechococcus sp. PCC 7002
Shewanella sp. W3-18-1

overall_design: We employed controlled cultivation and next-generation sequencing technology to identify transcriptional responses of euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and a marine facultative aerobe Shewanella putrefaciens W3-18-1 to investigate the effect of C sources and C flux directions on the interactions between these organisms.

first: Alex

middle: S

last: Beliaev

first: Margie

middle: F

last: Romine

first: Margrethe

last: Serres

first: Hans

middle: C

last: Bernstein

first: Bryan

middle: E

last: Linggi

first: Nancy

middle: G

last: Insern

first: William

middle: B

last: Chrisler

first: Leo

middle: A

last: Kucek

first: Eric

last: Hill

first: Grigoriy

last: Pinchuk

first: Donald

last: Bryant

first: H. Steven

last: Wiley

middle: K

last: Fredrickson

first: Allan

last: Konopka


title: ChIP-Seq analysis of PU.1, BATF, and JunB in murine BMDCs

status: Public on Dec 22 2013

city: South San Francisco

first: Jason

last: Hackney

state: CA

address: 1 DNA Way

department: Department of Bioinformatics and Computational Biology

institute: Genentech, Inc.

summary: Purpose: The purpose of this study is to identify the genome-wide binding sites for IRF4 interaction partners PU.1, BATF, and JunB in dendritic cells. These ChIP-seq data were integrated with gene expression analysis in IRF4-sufficient and -deficient BMDCs in order to assemble an IRF4 gene regulatory network.

overall_design: Hematopoietic bone marrow progenitors from C57BL/6 mice were differentiated with GM-CSF and IL-4 for 5 days. On day 6, BMDCs were stimulated for 6 hours with 100ng/ml LPS. Fixed chromatin was immunoprecipitated with anti-PU.1, BATF, and JunB antibodies and subjected to high-throughput sequencing. The sequencing data for the input DNA was previously submitted as GSM999807.

first: Zora

last: Modrusan

last: Vander Lugt

first: Aly

last: Khan

first: Smita

last: Agrawal

first: Justin

last: Lesch

first: Meijuan

last: Zhou

first: Wyne

middle: P

last: Lee

first: Summer

last: Park

first: Min

last: Xu

last: DeVoss

first: Chauncey

middle: J

last: Spooner

first: Cecile

last: Chalouni

first: Lelia

last: Delamarre

first: Ira

last: Mellman

first: Harinder

last: Singh


title: Regulation of NUMB alternative splicing by RBM5, RBM6 and RBM10 controls cancer cell proliferation (CLIP-Seq)

city: Budapest

first: Endre

last: Sebestyén

address: Üllői út 26.

department: 1st Department of Pathology and Experimental Cancer Research

country: Hungary

institute: Semmelweis University

summary: RBM5, a regulator of alternative splicing of apoptotic genes, and its close homologues, RBM6 and RBM10, are RNA binding proteins frequently deleted or mutated in lung cancer. We report that RBM5/6 and RBM10 antagonistically regulate the proliferative capacity of cancer cells and display distinct positional effects in alternative splicing regulation. We identify the Notch pathway regulator NUMB as a key target of these factors in the control of cell proliferation. NUMB alternative splicing, which is frequently altered in lung cancer, can regulate colony and xenograft tumor formation and its modulation recapitulates or antagonizes the effects of RBM5, 6 and 10 in cell colony formation. RBM10 mutations identified in lung cancer cells disrupt NUMB splicing regulation to promote cell growth. Our results reveal a key genetic circuit in the control of cancer cell proliferation.

overall_design: CLIP-Seq analysis of RBM5, RBM6 and RBM10, with 2 biological replicates and one non-specific control for each protein.

first: Elias

last: Bechara

first: Isabella

last: Bernardis

first: Eduardo

last: Eyras

first: Juan

last: Valcárcel


title: Genome-wide maps of transcriptional factor binding in mouse erythroid cells.

first: Jim

last: Hughes

summary: Regions bound by the erythroid transcription factors GATA1 and NFE2 and the general factor CTCF were identified in mouse erythroid cells using ChIP-seq.

overall_design: Chip-seq experiements were performed in mouse erythroid Ter119+ cells.

first: Deborah

last: Hay

first: Magnus

last: Lynch

first: Doug

last: Higgs


title: Transcriptional programming of dendritic cells for enhanced major histocompatibility complex class II antigen presentation

first: Jason

last: Hackney

sample_organism:
Homo sapiens
Mus musculus


title: TP63 regulation of epithelial to mesenchymal transition in an experimental prostate cell model

city: hangzhou

first: jie

last: liu

address: yuhangtang road 866

country: China

institute: zcni

summary: p63 is a transcription factor central for epithelial homeostasis and development. In our model of epithelial to mesenchymal transition (EMT) in a human prostate cell culture model, p63 was one of the most down-regulated transcription factors during EMT. We therefore investigated the role of p63 in EMT by a gain and loss of function approach. Over-expression of the predominant epithelial isoform DNp63a in mesenchymal EPT1B8 cells led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). This was corroborated by a reciprocal effect when p63 was knocked down in epithelial EP156T cells. Global gene expression analyses found that DNp63a induced gene modules involving cell adhesion genes in mesenchymal like cells. Genome-wide analysis of p63 binding sites by ChIP-seq analyses confirmed binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells.CDH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knock-down of these factors, respectively, were not sufficient alone or in combination with DNp63a to reverse the mesenchymal phenotype in EPT1 cells. The partial reversion of epithelial to mesenchymal transition might reflect the ability of DNp63a, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The utility of DNp63a expression and the potential of reduced EMP in order to counteract metastasis warrant further investigation.

overall_design: Examination of p63 binding profile in prostate cell model EP156T with EPT1 as negative control.

first: Karl-Henning

last: Kalland

first: Jan

middle: R

last: Olsen


title: Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells

city: Los Angeles

first: Martin

last: Martin

department: Pediatrics Gastroenterology

institute: University of California Los Angeles

summary: Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.

overall_design: Two samples of intestinal subepithelial myofibroblasts were used in this study.

first: Nanye

last: Lei

first: Ziyad

last: Jabaji

first: Jiafang

last: Wang

first: Vaidehi

last: Joshi

first: Garrett

last: Brinkley

first: Hassan

last: Khalil

first: Fengchao

first: Artur

last: Jaroszewicz

first: Matteo

last: Pellegrini

first: Linheng

last: Li

first: Michael

last: Lewis

first: Matthias

last: Stelzner

first: James

last: Dunn


title: Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain (RNA-seq)

city: Cambridge

first: Junjie

last: Guo

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute

summary: DNA methylation plays critical roles in the nervous system and has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes. Here we show that the single-base resolution neuronal DNA methylome from the adult mouse dentate gyrus consists of both CpG (~75%) and CpH (~25%) methylation (H = A/C/T). Neuronal CpH methylation is conserved in human brains, enriched in low CpG-density regions, depleted at protein-DNA interaction sites, and anti-correlated with gene expression. Functionally, both mCpGs and mCpHs can repress transcription in vitro and are recognized by MeCP2 in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNMT3A for active maintenance in post-mitotic neurons. These characteristics of CpH methylation suggest a significantly expanded proportion of the neuronal genome under cytosine methylation regulation and provide a new framework for understanding the roles of this key epigenetic modification in neuronal identity, maturation, plasticity and neurological disorders.

overall_design: Three biological replicates (dentate gyrus samples from C57Black6 mice) were analyzed by mRNA-seq


title: Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain

type: Methylation profiling by high throughput sequencing


title: Age-related methylation changes are associated with altered transcriptional circuitry [Methyl-seq]

city: Heidelberg

first: Guenter

last: Raddatz

address: Im Neuenheimer Feld 580

country: Germany

institute: German Center for Cancer Research

summary: Altered DNA methylation patterns represent an attractive mechanism for the phenotypic changes associated with human aging. Several studies have described age-related methylation changes to various extents, but their functional significance has remained largely unclear. We have now used an integrated methylome and transcriptome sequencing approach to characterize age-related methylation changes in the human epidermis and to analyze their impact on gene expression. Our results show limited and localized methylation differences between young and old methylomes at single-base resolution. Similarly, the comparison of transcriptomes from young and old samples revealed a highly defined set of differentially expressed genes with functional annotations in skin homeostasis. Further data analysis showed a robust correlation between age-related promoter hypermethylation and gene silencing, particularly at promoters that were pre-marked with stem cell-specific chromatin features. In addition, we also observed age-related methylation changes at transcription factor binding sites, with a significant enrichment of stem cell regulatory networks. Our results provide a high-resolution analysis of age-related methylation changes and suggest that they result in highly defined alterations in the transcriptional programme of the human epidermis. Interestingly, several of our findings can be interpreted to reflect epigenetic changes in aging stem cells, thus supporting a critical role of stem cells in human aging.

overall_design: Whole genome methylation analysis of H. sapiens. Two samples were analyzed, one sample containing DNA from young, one sample containing DNA from old human skin.

first: F

last: Lyko

first: M

last: Winnefeld


title: Small RNA profiling in an Arabidopsis transgenic line overexpressing AtmiR472

status: Public on Dec 19 2013

city: PARIS

first: Alexis

last: Sarazin

address: 46 rue d'Ulm

department: Ecole Normale Supérieure, Département de Biologie

country: France

institute: CNRS UMR 8186 - Biologie Moléculaire des Organismes Photosynthétiques

type: Non-coding RNA profiling by high throughput sequencing

summary: Virus resistance relies in some plant-viral interactions on the RNA-DEPENDANT RNA POLYMERASE 6 (RDR6), a major actor of RNA silencing that acts at the post-transcriptional level. Here, we demonstrate that RDR6 also plays a role in basal defense and race-specific resistance. RDR6 and the microRNA miR472, which target the mRNAs of disease resistance genes of coiled-coil nucleotide-binding leucine-rich-repeats family (e.g. RPS5), act in cooperation to control post-transcriptionally these immune receptors. Induction of these resistance genes is primed in rdr6- and miR472-elicited mutants and this effect is associated with an enhanced basal and race-specific immunity in these backgrounds.

overall_design: Comparison of Col0 WT sRNA with miR472 over-expressor mutant.

first: Martine

last: Boccara

first: Odon

last: Thiébeauld

first: Florence

last: Jay

first: Olivier

last: Voinnet

first: Lione

last: Navarro

first: Vincent

last: Colot


title: A genome-wide functional investigation into the roles of differentially expressed genes and microRNAs, including 24 transcription factor families, in tobacco roots during the drought stress response

city: Wenjiang,Chengdu

first: hongjun

last: liu

state: Sichuan

address: 211 Huimin Road

institute: Sichuan Agricultural University

summary: Drought stress response is a complex trait regulated at multiple levels. In the past few years, molecular and genomic studies have shown that several drought responsive genes (DRGs) with various functions are induced by drought stresses, and that various transcription factors (TFs) are involved in the regulation of stress-inducible genes. In addition to those DRGs mentioned above, microRNAs (miRNAs) are important regulators of gene expression at the posttranscriptional level by repressing mRNA expression. There is a complex interplay between transcriptional and post-transcriptional regulation of drought response that has not been extensively characterized in tobacco. In order to fully understand DRGs (including TFs) and different roles of miRNAs involved in the stress response, we sequenced and analysed three Digital Gene Expression (DGE) libraries in roots from drought treated tobacco plants, and four small RNA populations in roots, stems and leaves from control or drought treated tobacco plants. We identified 276 candidate DRGs in tobacco with sequence similarities to 64 known DRGs from model plants and crops and about 40% were TFs including WRKY, NAC, ERF and bZIP families. Furthermore, Out of these tobacco DRGs, 54differentially expressed DRGs included 21 TFs, which belonged to 24 TFs families such as NAC (6), MYB (4), ERF (10) and bZIP (1). Additionally, we confirmed expression of 39 known miRNA families (122 members) and five conserved miRNA families, which showed differential regulation under drought stress. Targets of miRNAs were further surveyed based on a recently published study, in which ten targets were DRGs. Finally, an integrated gene regulatory network has been proposed for the molecular mechanisms of the response of tobacco roots to drought stress using differentially expressed DRGs, the changed expression proﬁles of miRNAs and their target transcripts as a basis base on previous studies. In general, our data provide valuable information for future studies of the molecular mechanisms underlying tobacco roots in response to drought resistance in tobacco and other plants.

overall_design: Three tobacco (Nicotiana tabacum L.) roots tag-based DGE libraries treated at three time points (0, 6 and 48 h) with 20% PEG6000 were generated using Illumina 1G technology. The samples for four small RNA libraries was used based on the result of physiological index measurement as follows: equal quantities (10 ug) of total RNA isolated from tobacco roots treated with two time points (6 and 48 h) were mixed together to construct the drought -treated small RNA library (Root-treat), and total RNA prepared from the control roots sample was used to construct the control small RNA library (Root-ck). In addition, we also constructed another two libraries (stem and leaf) from leaves and stems of control plants, respectively. These libraries were constructed using the Small RNA Sample Prep Kit (Illumina, San Diego, CA) and then sent for Solexa sequencing.

first: Fuqiang

last: Yin

first: Hongjun

last: Liu

first: Wenyou

last: Zhang

first: Cheng

last: Qin

first: Xinhui

last: Liao

first: Ming

first: Aiguo

last: Yang

first: Zhenhua

last: Wang

first: Zhiming

first: Yaou

last: Shen

first: Jian

last: Gao

first: Chenggang

last: Luo

first: Haobao

first: Thomas

last: Lübberstedt

first: Guangtang

last: Pan


title: Quantitative single-cell RNA-seq

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 2

department: MBB

country: Sweden

institute: Karolinska Institutet

summary: Purpose: We applied cDNA molecule counting using unique molecular identifiers combined with high-throughput sequencing to study the transcriptome of individual mouse embryonic stem cells, with spike-in controls to monitor technical performance. We further examined transcriptional noise in the embryonic stem cells.

overall_design: One 96-well plate of single-stranded cDNA libraries generated from 96 single R1 mouse embryonic stem cells sequenced on two lanes, and one 96-well plate of the same libraries further amplified by 9 PCR cycles sequenced on one lane.

first: Saiful

last: Islam

first: Amit

last: Zeisel

first: Peter

last: Lönnerberg

first: Simon

last: Joost

first: Gioele

last: La Manno

first: Pawel

last: Zajac


title: 3P-seq analysis of S. cerevisiae

city: San Francisco

first: David

last: Weinberg

address: 600 16th Street, GH S572F

institute: University of California, San Francisco

summary: PolyA Position Profiling (3P-seq) for S. cerevisiae

overall_design: Analysis of S. cerevisiae

last: Bartel


title: Cell cycle positioning drives heterogeneity within the pluripotent stem cell compartment

city: Athens

first: Stephen

last: Dalton

state: GEORGIA

address: 500 D.W. Brooks drive Athens G

department: Biochemistry & Molecular Biology

institute: University of Georgia

summary: Heterogeneity in pluripotent cells marks a metastable state where cells may drift between native and lineage-primed populations.  While the role for these heterogeneities are unclear, they may reflect the dynamic equilibriums of signaling networks and have a direct effect on differentiation potentialities.  Here, we report the role of the cell cycle in establishing heterogeneity of human pluripotent stem cells.  By utilizing the FUCCI cell cycle indicator system coupled to fluorescent activated cell sorting (FACS), we have uncovered that the cell cycle drives heterogeneity at the epigenetic, transcriptional and post-transcriptional levels.  Our data show widespread dynamics in 5-hydroxymethylcytosine (5hmC) during the cell cycle.  Furthermore, transcript profiling by RNA-sequencing identified >500 genes that were cell cycle-regulated, of which the largest cohort of genes were transcriptional regulators. In sum, we demonstrate the role of the cell cycle in coordinating cellular transitions between metastable states in pluripotent stem cells.

overall_design: mRNA sequencing of the cell cycle phases; early & late G1, S and G2/S from human ES cells in triplicate.

first: Amar

middle: M

last: Singh

first: James

last: Chappell


title: Translation rates and 3' ends of *S. cerevisiae* genes


title: Ribosome profiling study of dom34 and hbs1 knockout strains using short (16-nt) and long (28-nt) monosome-protected footprints and disome-protected footprints

city: Bethesda

first: Nicholas

last: Guydosh

state: MD

address: 8 Center Dr, Bldg 8, 220

department: NIDDK

institute: National Institutes of Health

summary: Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here we use ribosome profiling methodology to reveal a high- resolution molecular characterization of Dom34 function in vivo. We show that Dom34 removes stalled ribosomes from mRNAs that are truncated but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3 ́ UTRs. These ribosomes appear to gain access to the 3 ́ UTR via a mechanism that does not require decoding of the mRNA. These results suggest that Dom34 carries out the important task of rescuing ribosomes found in noncoding regions.

overall_design: 25 samples are included in the study (2 mRNA-Seq samples and 23 ribosome footprint profiling samples). These include wild-type and dom34 or hbs1 knockout strains that were created in a variety of genetic backgrounds, treated with various agents in cell culture (e.g. diamide, 3-AT, or glucose starvation), treated differently during cell lysis (use of cycloheximide vs. other ribosome-stabilizing agents), or prepared in different ways after cell lysis (e.g. retention of short vs. long monosome-protected footprints or disome footprints).

first: Rachel

last: Green


title: Determinants of ribosome density in yeast

summary: Here, we use ribosome-footprint profiing and mRNA-seq to determine the average ribosome density on each gene in S. cerevisiae. We then perform quantitative modeling to identify the molecular determinants of ribosome density.

middle: W

last: Eichhorn


title: Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans

city: Worcester

first: Craig

last: Mello

address: 368 Plantatoin Street, Suite AS5-2047

department: Program in Molecular Medicine

institute: University of Massachusetts Medical School

summary: During each life cycle germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute (AGO) proteins and their small-RNA cofactors are expressed during male gametogenesis and promote male fertility. Here we show that the CSR-1 AGO functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal that represents an epigenetic memory of male-specific gene expression, while CSR-1, which is abundant in mature sperm, transmits this memory to offspring.  Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small-RNA complexes that constitute a memory of which genes were active in preceding generations.

overall_design: Examine small RNA changes in WT and alg-3/4 mutant males cultured at 20°C and 25°C, as well as determine the small RNAs enriched in a FLAG::CSR-1 IP from male worms grown at 25°C.  mRNA sequencing was also performed to determine how transcripts targeted by small RNAs change in mutant background at 20°C and 25°C.

first: Colin

last: Conine

last: Moresco

first: Weifeng

last: Gu

first: Masaki

last: Shirayama

first: Darryl

last: Conte

first: John

last: Yates III


title: Rapid and Pervasive Changes in Genome-Wide Enhancer Usage During Mammalian Development

city: Berkeley

first: Alex

last: Nord

address: 1 Cyclotron Road, Mail Stop 84-171

department: Genomics Divsion

institute: Lawrence Berkeley National Laboratory

summary: Here we used epigenomic profiling for H3K27ac, a mark of active enhancers to examine the genome-wide in vivo utilization of enhancers in three different mouse tissues across seven developmental stages, ranging from mid-gestation through adulthood. The majority of the ~90,000 enhancers identified exhibited tightly temporally restricted activity windows and were associated with stage-specific biological functions and regulatory pathways in individual tissues. Comparative genomic analysis revealed that evolutionary conservation of enhancers decreases following mid-gestation across all tissues examined. The dynamic enhancer activities uncovered in this study illuminate rapid and pervasive temporal in vivo changes in enhancer usage underlying developmental processes and demonstrate the value of time-course chromatin profiling of relevant tissues across development.

overall_design: Examination of H3K27ac in mouse forebrain, heart and liver tissues collected across developmental stages.

first: Axel

last: Visel

first: Len

last: Pennacchio


title: RNAseq of PRMT4KD in human cord blood derived CD34+ cells

city: New York

first: Bic

last: MSKCC

state: NY

address: 1275 York Ave.

institute: Memorial SLoan-Kettering Cancer Center

summary: Defining the role of epigenetic regulators in normal hematopoiesis has become critically important, as recurrent mutations or aberrant expression of these genes has been identified in both myeloid and lymphoid hematological malignancies. We have found that PRMT4, a type I arginine methyltransferase, whose function in normal and malignant hematopoiesis is unknown, is overexpressed in AML patient samples. In support of an oncogenic role for PRMT4, we find that its overexpression blocks the myeloid differentiation of human stem/progenitor cells (HSPCs) while its knockdown (KD) is sufficient to induce myeloid differentiation of HSPCs and multiple AML cell lines. Although classically thought of as a co-activator, we found that PRMT4 functions to repress the expression of miR-223 in HSPCs via the methylation of RUNX1, which triggers the assembly of a multi-protein repressor complex that includes DPF2. As part of a feedback loop, PRMT4 expression is repressed post-transcriptionally by miR-223 during the normal differentiation process. These data reveal an unidentified role of PRMT4 in myeloid differentiation and its unexpected repressive role in transcriptional regulation. Furthermore, depletion of PRMT4 results in the differentiation of myeloid leukemia cells in vitro and their decrease proliferation in vivo. Thus, targeting PRMT4 holds potential as a novel therapy for acute myelogenous leukemia.

overall_design: Purified human primary CD34+ cells were transduced with lentiviruses carrying PRMT4KD or scramble control shRNAs. Total RNA was extrated. RNAseq was performed to identify target genes that are regulated by PRMT4. Experiments were performed in triplicate.

first: Ly

last: Vu

last: Nimer


title: Genome wide co-localization of Polycomb orthologs and their effects on gene expression in human fibroblasts

city: London

first: Gordon

last: Peters

address: 44 Lincolns Inn Fields

institute: CRUK London Research Institute

summary: We have identfied antibodies that support chromatin immunoprecipitation (ChIP) of 11 components of Polycomb repressive complex 1 (PRC1) and shown that multiple variants of PRC1 associate with the same DNA.  By performing ChIP-seq with antibodies against CBX6, CBX7, CBX8, RING1 and RING2, in two strains of human fibroblasts, we find that  all five proteins co-localize at multiple sites in the genome. The binding profiles have inherent architecture that is conserved between the two strains, but there are also a substantial number of differences between the strains, possibly reflecting their anatomical position. The presence of PcG proteins does not equate with gene silencing and their genomic position does not change at senescence.

overall_design: Chromatin immunoprecipitation and sequencing (ChIP-seq) in two strains of  human fibroblasts using antibodies against three Pc orthologs (CBX6, CBX7 and CBX8) and both Psc orthologs (RING1 and 2). Validation by ChIP-PCR and re-ChIP.  Strand specific RNA-seq and validation by qRT-PCR.

first: Helen

last: Pemberton

first: Emma

last: Anderton

first: Harshil

last: Patel

first: Sharon

last: Brookes

first: Richard

last: Palermo

first: Julie

last: Stock

first: Marc

last: Rodriguez-Niedenführ

first: Tomas

last: Racek

first: Lucas

last: de Breed

first: Aengus

last: Stewart

first: Nik

last: Matthews


title: RNA-seq identifies novel transcript elements and transcript processing in the normal and failing hearts

city: Rochester

first: LIGUO

last: WANG

state: MN

address: 200 1st St SW

department: Division of Computational Biology

institute: Mayo Clinic

summary: Changes in gene expression contribute to the pathogenesis of heart failure. The sequence, expression level, and structure of the human cardiac transcriptome are incompletely described, as are their changes in heart disease. High throughput transcriptome sequencing (RNA-seq) is a quantitative and unbiased approach to measure transcript level and to identify novel transcribed elements or transcript splicing. Here we acquired 975.2 x 106 mapped RNA-seq reads in 15 control and 15 ischemic cardiomyopathy (ICM) hearts, obtained at the time of heart transplantation. We identified over 1000 differentially expressed transcripts, and thousands of novel transcribed elements, some of which were differentially expressed in between control and ICM groups. We found that transcript processing of several cardiac genes was deranged in ICM. For instance, the ratio between specific MYH6 exons was significantly changed in ICM compared to controls, while this type of inter-exon variation was not observed for the adjoining gene MYH7. This RNA-seq study of the human heart failure transcriptome revealed the diversity of transcripts expressed in the human heart and their complex patterns of expression in the diseased heart.

overall_design: Transcriptome profiling (RNA-seq) of 15 control and 15 ischemic cardiomyopathy (ICM) hearts using Illumina GAII and SOLiD

first: Liguo

last: Wang

first: Yongwu

last: Hu

first: William

last: Pu


title: Switch Enhancer Elements Control Cell Fate in Human Embryonic Stem Cells

status: Public on Dec 18 2013

city: Toronto

first: Tobias

middle: Andreas

last: Beyer

state: ONT

address: 600 University Ave

department: Lunenfeld-Tannenbaum Research Institute

institute: Mount Sinai Hospital

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

overall_design: Refer to individual Series


title: The SLE transcriptome exhibits evidence of chronic endotoxin exposure and has widespread dysregulation of non-coding and coding RNAs

city: Philadelphia

first: Kathleen

middle: E.

last: Sullivan

state: PA

address: 3615 Civic Center Blvd.

department: Immunology

institute: The Children's Hospital of Philadelphia

summary: We utilized next generation sequencing of transcripts (RNA-seq) to characterize the SLE transcriptome in monocytes.This is the first RNA-seq study of SLE using clinical subjects and we found a transcriptome that exhibits quantitative alterations as defined by the level of gene expression but also qualitative differences with widely altered splicing preference and non-coding RNA transcription.

overall_design: total RNA from 8 heathy controls and 9 SLE patients were used to RNA sequence and comparision the whole transcriptome alteration

first: K

first: L

last: Shi

first: Z

last: Zhang


title: Salmonella enterica serovar Typhimurium 4/74 transcriptomics using strand-specific RNA-seq

city: Liverpool

first: Jay

last: Hinton

address: Crown Street

department: Institute of Integrative Biology

institute: University of Liverpool

summary: Bacterial transcription networks typically consist of hundreds of transcription factors and thousands of promoters. However, current attempts to map bacterial promoters have failed to report the true complexity of bacterial transcription. The differential RNA-seq (dRNA-seq) approaches only identified a subset of promoters because they involved few growth conditions. Here, we present a simplified approach for global promoter identification in bacteria, based upon the analysis of RNA-seq data from multiple environmental conditions. RNA was extracted from Salmonella enterica serovar Typhimurium (S. Typhimurium) grown in 22 different environmental conditions, which were devised to reflect the pathogenic lifestyle of S. Typhimurium. Individual RNA samples were combined into two pools for sequencing. In just two runs of strand-specific RNA-seq and dRNA-seq of the pooled sample we identified 3701 promoters (Pool sample). In further experiments, we found that individual in vitro conditions stimulate the expression of about 60% of the S. Typhimurium genome, whereas the suite of 22 conditions induced expression of 87% of S. Typhimurium genes. We discovered environmental conditions that induce many genes within Salmonella pathogenicity islands and identified 78 new sRNAs. In S. Typhimurium there is now experimental evidence for 280 sRNAs, and we classified them in terms of location and Hfq-binding.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. ST4/74

overall_design: Transcriptome analysis of S. Typhimurium 4/74 using RNA from 22 different conditions using RNA-seq. Also, RNA from each condition was pooled into one sample (RNA Pool). Differential RNA-seq (dRNA-seq) was performed for 5 of the samples from the 22 environmental conditions.

first: Carsten

last: Kröger

first: Aoife

last: Colgan

first: Shabarinath

last: Srikumar

first: Kristian

last: Händler

first: Sathesh

last: Sivasankaran

first: Disa

last: Hammarlöf

first: Rocío

last: Canals

first: Joe

last: Grissom

first: Tyrrell

last: Conway

first: Karsten

last: Hokamp


title: Transcriptomic analyses of Myc and xmrk induced zebrafish liver cancer

status: Public on Dec 17 2013

city: Singapore

first: Weiling

last: Zheng

address: Science Drive 4

country: Singapore

institute: National University of Singapore

summary: To compare the characteristics and mechanisms of Myc and xmrk induced zebrafish liver tumor, next generation sequencing-based SAGE analyses were used to examine the transcriptomes of tumor and control samples. The results indicated that relatively small overlaps of significantly deregulated genes and biological pathways among different zebrafish liver tumor models.Nevertheless, they all significantly correlate with advanced or very advanced human hepatocellular carcinoma (HCC). Molecular signature from each oncogene-induced zebrafish liver tumor correlated with only a small subset of human HCC samples, and they share conserved up-regulated pathways. A short list of commonly deregulated genes among different zebrafish liver tumors showed accordant deregulation in the majority of human HCCs, suggesting that they may serve as common diagnosis markers and therapeutic targets.Thus, these transgenic zebrafish models with well-defined oncogene-induced tumors are valuable tools for molecular classification of human HCCs and for understanding of molecular drivers in hepatocarcinogenesis in each human HCC subgroup.

overall_design: Transcriptome profiling of Myc tumor sample (X-M+D+) and control samples (X-M-D-, X-M+D-, X-M-D+), xmrk tumor sample (X+M-D+) and control samples (X-M-D-, X+M-D-, X-M-D+), were generated by deep sequencing, using 3' RNA-SAGE on SOLiD system

first: Zhen

last: Li

first: Zhiyuan

last: Gong


title: DNA Methyltransferase inhibition reverses epigenetically embedded phenotypes in lung cancer preferentially affecting Polycomb target genes

first: Christian

last: Rohde

address: Im Neuenheimer Feld 410

institute: Heidelberg University

summary: Cancer cell phenotypes are partially determined by epigenetic specifications such as DNA methylation. Metastasis development is a late event in cancerogenesis and might be associated with epigenetic alterations. Here, we analyzed genome wide DNA methylation changes that were associated with pro-metastatic phenotypes in non-small cell lung cancer with Reduced Representation Bisulfite Sequencing. DNMT-inhibition by 5-Azacytidine at low concentrations reverted the pro-metastatic phenotype. 5-Azacytidine led to preferential loss of DNA methylation at sites that were DNA hypermethylated during the in vivo selection. Changes in DNA methylation persisted over time.

last: Müller-Tidow


title: mRNA expression profiles of WT and MLL4-/- after MyoD-induced myogenesis

first: Kai

last: Ge

address: 50 South Dr Rm 4154

institute: NIH

summary: Enhancers play a central role in cell-type-specific gene expression and are marked by H3K4me1/2. Active enhancers are further marked by H3K27ac. However, the methyltransferases responsible for the deposition of H3K4me1/2 on enhancers remain elusive. Furthermore, the functions of these methyltransferases on enhancers and associated cell-type-specific gene expression are poorly understood. Here, we identify MLL4 (KMT2D) as a major H3K4 mono- and di-methyltransferase in mammalian cells. Using adipogenesis and myogenesis as model systems, we show that MLL4 exhibits cell-type- and differentiation-stage-specific genomic binding and is predominantly localized on enhancers. MLL4 co-localizes with lineage-determining transcription factors (TFs) on active enhancers during differentiation. Deletion of MLL4 dramatically decreases H3K4me1/2 and H3K27ac on enhancers and leads to severe defects in cell-type-specific gene expression and cell differentiation. Finally, we provide evidence that lineage-determining TFs recruit and require MLL4 to establish enhancers critical for cell-type-specific gene expression. Together, these results identify MLL4 as an H3K4 mono-/di-methyltransferase required for enhancer activation during cell differentiation.

overall_design:
RNA-Seq analysis of mRNA profiles in adenoviral GFP- or Cre-infected MLL3-/-;MLL4-flox/flox cells.
Preadipocytes: brown preadipocytes before differentiation.
D5 myocytes: 5 days after MyoD-induced myogenesis of brown preadipocytes.

first: Ji-Eun

last: Lee

first: Chaochen

last: Wang

first: Shiliyang

last: Xu

first: Weiqun

last: Peng


title: Examination of genome-wide methylation changes in lung cancer cell lines A549 (A) and HTB56 (H) [RRBS-Seq experiments]

summary:
Cancer cell phenotypes are partially determined by epigenetic specifications such as DNA methylation. Metastasis development is a late event in cancerogenesis and might be associated with epigenetic alterations. Here, we analyzed genome wide DNA methylation changes that were associated with pro-metastatic phenotypes in non-small cell lung cancer with Reduced Representation Bisulfite Sequencing. DNMT-inhibition by 5-Azacytidine at low concentrations reverted the pro-metastatic phenotype. 5-Azacytidine led to preferential loss of DNA methylation at sites that were DNA hypermethylated during the in vivo selection. Changes in DNA methylation persisted over time.

Keywords: Methylation profiling by high throughput sequencing

overall_design: We studied genome-wide methylation changes in lung cancer cell lines A549 (A) and HTB56 (H). We generated NSCLC lines with highly increased propensity to form tumor nodules in murine lungs after intravenous injections. In addition to the normal cell lines (0R) we analyzed the methylome of the the cell lines after three rounds of in vivo selection towards a highly metastatic phenotype (3R). Next we studied changes in the methylome of highly metastatic cell lines after DNA Methyltransferase inhibition by 5-Azacytidine treatment at low concentrations (250 nM & 1000 nM) for 6 days. During treatment cells were supplemented with fresh medium every 48 hours. After 6 days of 5-Azacytidine exposure, cells were washed three times with PBS to wash out the drug. The cells were released for additional 7 days in regular medium. We followed up the DNA methylation changes at day 13 of the experiment.

first: Carsten


title: Genome-wide maps of MyoD, MLL4 and histone modifications during MyoD-induced myogenesis in WT and MLL4-/- brown preadipocytes

overall_design:
ChIP-Seq of MyoD, MLL4 and histone modifications (H3K4me1, H3K4me3, and H3K27ac) in adenoviral GFP- or Cre-infected MLL3-/-;MLL4-flox/flox cells.
Preadipocytes: brown preadipocytes before differentiation.
D5 myocytes: 5 days after MyoD-induced myogenesis of brown preadipocytes.


title: Switch Enhancer Elements Control Cell Fate in Human Embryonic Stem Cells (ChIP-Seq)

first: Tobias

last: Beyer

summary: A small toolkit of morphogens is used repeatedly to direct development, raising the question of how context dictates interpretation of the same cue. One example is the TGFβ pathway that in human embryonic stem cells fulfills two opposite functions: pluripotency maintenance and mesendoderm (ME) specification. Using proteomics coupled to analysis of genome occupancy, we uncover a regulatory complex comprised of transcriptional effectors of the Hippo pathway (TAZ/YAP/TEAD), the TGFβ   pathway (SMAD2/3) and the pluripotency regulator OCT4 (TSO). TSO collaborates with NuRD repressor complexes to buffer pluripotency gene expression, while suppressing ME genes. Importantly, the SMAD DNA binding partner FOXH1, a major specifier of ME, is found near TSO elements and upon fate specification we show that TSO is disrupted with subsequent SMAD-FOXH1 induction of ME. These studies define switch enhancer elements and provide a framework to understand how cellular context dictates interpretation of the same morphogen signal in development.

overall_design: ChIP experiment, a total of 8 samples: 4 samples DMSO treated (Input ctr, IgG ctr, SMAD2 precipitation, TEAD4 precipitation), 4 samples SB431542 (Input ctr, IgG ctr, SMAD2 precipitation, TEAD4 precipitation)

first: Jeffrey

middle: L

last: Wrana


title: Genome-wide maps of C/EBPbeta, MLL4 and histone modifications in C/EBPbeta-overexpressed WT and MLL4-/- brown preadipocytes

overall_design: ChIP-Seq analyses of C/EBPbeta, MLL4 and histone modifications (H3K4me1, H3K27ac) in vec- or C/EBPbeta-overexpressing, adenoviral GFP- or Cre-infected, MLL3-/-MLL4-flox/flox brown preadipocytes without induction of differentiation.


title: Genome-wide maps of adipogenic TFs during brown preadipocyte differentiation

overall_design: ChIP-Seq analyses of adipogenic TF (C/EBPalpha, C/EBPbeta, and PPARgamma) and Pol II profiles at D0 (day 0) and D2 (day 2) of adipogenesis in WT (MLL3-/-) and MLL4 KO (MLL3-/-;MLL4-/-) brown preadipocytes.


title: Age-related methylation changes are associated with altered transcriptional circuitry [RNA-seq]

first: Guenter

last: Raddatz

address: Im Neuenheimer Feld 580

institute: German Center for Cancer Research

summary: Altered DNA methylation patterns represent an attractive mechanism for the phenotypic changes associated with human aging. Several studies have described age-related methylation changes to various extents, but their functional significance has remained largely unclear. We have now used an integrated methylome and transcriptome sequencing approach to characterize age-related methylation changes in the human epidermis and to analyze their impact on gene expression. Our results show limited and localized methylation differences between young and old methylomes at single-base resolution. Similarly, the comparison of transcriptomes from young and old samples revealed a highly defined set of differentially expressed genes with functional annotations in skin homeostasis. Further data analysis showed a robust correlation between age-related promoter hypermethylation and gene silencing, particularly at promoters that were pre-marked with stem cell-specific chromatin features. In addition, we also observed age-related methylation changes at transcription factor binding sites, with a significant enrichment of stem cell regulatory networks. Our results provide a high-resolution analysis of age-related methylation changes and suggest that they result in highly defined alterations in the transcriptional programme of the human epidermis. Interestingly, several of our findings can be interpreted to reflect epigenetic changes in aging stem cells, thus supporting a critical role of stem cells in human aging.

overall_design: Whole transcriptome analysis of H. sapiens. Two samples were analyzed, one sample containing RNA from young, one sample containing RNA from old human skin.

first: F

last: Lyko

first: M

last: Winnefeld


title: Genome-wide maps of histone modification in WT and MLL4-/- during brown preadipocyte differentiation

overall_design: ChIP-Seq analyses of histone modifications (H3K4me1, H3K4me2, H3K4me3, and H3K27ac) at D0 (day 0) and D2 (day 2) of adipogenesis in WT (MLL3-/-) and MLL4 KO (MLL3-/-;MLL4-/-) brown preadipocytes.


title: Landscape and variation of RNA secondary structure across the human transcriptome

city: Stanford

first: Kun

last: Qu

address: 269 Campus Dr. CCSR 2150

department: Dermatology

institute: Stanford University

summary: In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. The ability of RNA to base pair with itself and other nucleic acids endow RNA with the capacity to form extensive structures, which are known to influence practically every step in the gene expression program1. Yet the nature of most RNA structures or effects of sequence variation on structure are not known.  Here we report the initial landscape and variation of RNA secondary structures (RSS) in a human family trio, providing a comprehensive RSS map of human coding and noncoding RNAs. We identify unique RSS signatures that demarcate open reading frames, splicing junctions, and define authentic microRNA binding sites.  Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over one thousand transcribed single nucleotide variants (~15% of all transcribed SNVs) alter local RNA structure; these “RiboSNitches”2 occur in disease-associated variants.  We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSS. Selective depletion of RiboSNitches versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3’UTRs, binding sites of miRNAs and RNA binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

overall_design:
RNA structure probing is performed at 37˚C on poly(A)+ selected RNAs from GM12878, GM12891 and GM12892 cell lines, as well as on native proteinized RNAs from GM12878.  The structure probed RNAs is then cloned into a sequencing library using modied Ambion RNA sequencing kit compatible with the Illumina platform.  The samples were deep sequenced using Illumina's Hi-Seq platform.
AGO CLIP was performed as reported. Cells were crosslinked with UV and lysed using published protocols. AGO2 was enriched using immunopurification. The RNA-protein complex was digested with ribonuclease and purified by gel electrophoresis. Purified RNA was reverse transcribed and cDNA molecules were amplified and sequenced as described.

first: Yue

last: Wan

first: Qiangfeng

last: Zhang

first: Ryan

last: Flynn

first: Ohad

last: Manor

first: Zhengqing

last: Ouyang

first: Jiajing

first: Michael

last: Snyder

first: Eran

last: Segal

first: Howard

middle: Y

last: Chang


title: mRNA expression profiles of WT and MLL4-/- during brown preadipocyte differentiation

first: Kai

last: Ge

overall_design: RNA-Seq analyses of mRNA profiles at D0 (day 0) and D2 (day 2) of adipogenesis in WT (MLL3-/-) and MLL4 KO (MLL3-/-;MLL4-/-) brown preadipocytes.

first: Ji-Eun

last: Lee

first: Chaochen

last: Wang

first: Shiliyang

first: Weiqun


title: Characterizing the TRb1 cistrome in mouse liver

city: Boston

first: Preeti

last: Ramados

address: 3 Blackfan Cir

institute: Beth Israel Deaconess Medical Center

summary: ChIP-seq data from mouse liver over-expressing GFP and fed a PTU diet and treated with saline, or overexpressing biotinylated TRb1 and GFP and fed a PTU diet, treated with either saline or T3

overall_design: Mouse liver was infected with adenovirus that either expressed only GFP or GFP and biotinylated TRb1 and were fed a PTU diet, followed by injections with only saline (GFP-only livers) or with saline or T3 (Biotinylated-TRb1 and GFP livers). ChIP assays were performed using streptavidin agarose to capture biotinylated TRb1 followed by library construction and sequencing.

first: P

last: Ramadoss

first: B

last: Abraham

first: L

last: Tsai

first: Y

last: Zhou

first: R

middle: H

last: Costa-e-Sousa

first: F

last: Ye

last: Bilban

first: K

last: Zhao

first: A

middle: N

last: Hollenberg


title: Genome-wide maps of MLL4 during brown preadipocyte differentiation

overall_design: ChIP-Seq analyses of MLL4 binding profiles at D0 (day 0), D2 (day 2), and D7 (day 7) of adipogenesis in WT (MLL3-/-) and MLL4 KO (MLL3-/-;MLL4-/-) brown preadipocytes. D0 and D2 input DNA samples can be found in GSE50417.

last: Xu

last: Peng


title: Silencing of odorant receptor gene expression by G protein βγ signaling ensures the expression of one odorant receptor per olfactory sensory neuron

status: Public on Dec 16 2013

city: Padova

first: Davide

last: Risso

address: Via C. Battisti, 241

country: Italy

institute: University of Padova

summary: Olfactory sensory neurons express just one out of a possible ~1000 odorant receptor genes, reflecting an exquisite mode of gene regulation. In one model, once an odorant receptor is chosen for expression, other receptor genes are suppressed by a negative feedback mechanism, ensuring a stable functional identity of the sensory neuron for the lifetime of the cell. The signal transduction mechanism subserving odorant receptor gene silencing remains obscure, however. Here we demonstrate in the zebrafish that odorant receptor gene silencing is dependent on receptor activity. Moreover, we show that signaling through G protein βγ subunits is both necessary and sufficient to suppress the expression of odorant receptor genes, and likely acts through histone methylation to maintain the silenced odorant receptor genes in transcriptionally inactive heterochromatin. These results provide new insights linking receptor activity with the epigenetic mechanisms responsible for ensuring the expression of one odorant receptor per olfactory sensory neuron.

overall_design: Total 6 samples were analyzed-3 controls & 3 samples

first: Todd

last: Ferreira

first: Sarah

last: Wilson

first: Yoon

last: Choi

first: Sandrine

last: Dudoit

first: Terence

last: Speed

first: John

last: Ngai


title: Identification of FUS RNA targets in HeLa cells

city: Winnipeg

first: Geoffrey

last: Hicks

state: Manitoba

address: BMSB 669, 745 Bannatyne Avenue

department: Biochemsity & Medical Genetics

institute: University of Manitoba, Manitoba Institue of Cell Biology

summary: We identified a landscape of FUS-binding RNA targets in HeLa cells.  The majority of the FUS binding sites are in introns of pre-mRNAs and less are in exons and untranslated regions. Significant FUS binding in introns flanking cassette exons, long intron (>100kb) containing transcripts and noncoding RNAs were detected in our study. We specifically determined the function of FUS in regulating the alternative splicing of cassette exons. The top FUS-associated cassette exon is exon 7 of the pre-mRNA of FUS itself. We demonstrated that FUS is a repressor of its own exon 7 splicing. FUS autoregulates its own protein levels by exon 7 alternative splicing and nonsense mediated decay. Moreover, Amyotrophic Lateral Sclerosis (ALS) linked FUS mutants are deficient in FUS autoregulation.

overall_design: CLIP-seq of FUS in HeLa cells

first: Yueqin

first: Songyan

last: Liu


title: Whole transcriptome profiling of the two major subtypes of diffuse large B cell lymphoma

first: Jennifer

middle: Marion

last: Hardee

state: California

address: 300 Pasteur Dr., M-344

summary: The goal of this study is to identify the transcriptome differences between the two major subtypes of diffuse large B cell lymphoma (DLBCL).  DLBCL is the most common form of non-Hodgkin’s lymphoma and has two major subtypes: germinal center B-cell-like (GCB) and activated B-cell-like (ABC).  When compared to the GCB form, ABC lymphomas respond much more poorly to current therapies. To investigate how gene expression changes might contribute to this aggressive phenotype, we have used RNA-Seq to profile the whole transcriptome in 8 DLBCL cell lines (4 GCB subtype, 4 ABC) that are derived from patient tumors.  1,545 genes are differentially expressed between subtypes (FDR < 0.05), approximately 7% of the transcriptome.  The vast majority of these genes (81%, n = 1251) are more highly expressed in the ABC cell lines.  In contrast, only 294 genes (19%) are more highly expressed in the GCB cell lines.  Half (n = 765) of the genes with greater ABC subtype expression demonstrate very low read counts (< 5) in the GCB cell types.  Conversely, only 21 genes that are more highly expressed in GCB are unique to that subtype.  The prevalence of such “on/off” genes indicates that the major differences between ABC and GCB DLBCL are due almost exclusively to additional gene expression in ABC, rather than the two subtypes having divergent but equally active genetic programs.

overall_design: Measurement and comparison of gene expression in 8 cell lines representing the 2 subtypes of DLBCL.  4 cell lines are subtyped as ABC and 4 are subtyped as GCB.  2 replicates are present for each cell line.  (Cell line OCI-Ly19 was not included in the analysis because its gene expression clustered in between the subtypes, probably due to its EBV+ status.  However, its sequencing runs have been included for completeness.)

first: Zhengqing

last: Ouyang

first: Yuping

last: Zhang

first: Anshul

last: Kundaje

first: Phillippe

last: Lacroute

first: Michael

last: Snyder


title: Genome wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

institute: Centre for Immunity, Infection and Evolution

summary: The protozoan parasites Trypanosoma brucei spp. are responsible for important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ?slender? forms and transmissible ?stumpy? forms that are quiescent, awaiting uptake in a tsetse fly bloodmeal. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. The response is triggered by an elusive ?stumpy induction factor? (SIF) whose intracellular signaling pathway is also completely uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains cannot respond to SIF, but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to cell permeable cAMP or AMP analogues to select cells that remained proliferative and so were unresponsive to these signals. Genome-wide ion torrent-based RNA interference Target sequencing (RIT-seq) identified a cohort of genes implicated in all steps of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified genes at each step have been validated in cells naturally capable of stumpy formation, confirming their role in SIF-induced density sensing and cellular quiescence.

overall_design: RNA was isolated from AnTat1.1 cells grown in mice with or without doxycycline. RBP7 (Tb927.10.12100) RNAi lines were grown in mice with or without doxycycline. Since the RBP7 RNAi is leaky (six independent cell lines were analysed and all were significantly leaky), transcripts that showed elevated or reduced abundance in both RNAi induced and uninduced populations were identified in comparison to the AnTat1.1 control.  A similar approach was adopted for the RBP7 over-expressing lines.

first: Binny

last: Mony

first: Keith

last: Matthews


title: Gene expression analysis of high-glucose stimulated human mesangial cells and tubular epithelial cells

city: Kanazawa

first: Yasunori

last: Iwata

state: Ishikawa

country: Japan

institute: Kanazawa University

summary: we performed genome-wide transcriptome profiling in mesangial cells and tubular epithelial cells (TECs), which were stimulated by high glucose (HG) and detected the expression of inflammation associated genes. HG increased the mRNA expression of oxidative stress, inflammasome and mammalian target of rapamycin (mTOR) related genes in mesangial cells. Pro-inflammatory/Th1 gene expression was upregulated, but Th2 related gene expression was downregulated in mesangial cells. In TECs, HG stimulation increased pro-inflammatory/Th1/Th2 gene expression.

overall_design: mRNA profiles of human mesangial cells and tubular epithelial cells, which were stimuated HG for 24 hours or mannitol contorol.

first: Y

first: K

last: Furuichi

first: S

last: Hashimoto

last: Yokota

first: H

last: Yasuda

first: N

last: Sakai

last: Kitajima

first: T

last: Toyama

last: Shinozaki

first: A

last: Sagara

last: Matsushima

last: Kaneko

last: Wada


title: Whole genome mapping of STAT3 binding sites in the two major subtypes of diffuse large B cell lymphoma

first: Jennifer

last: Hardee

summary: The goal of this study is to identify the effect of the transcription factor STAT3 in the two major subtypes of diffuse large B cell lymphoma (DLBCL).  STAT3 is a signal transducer that, when dysregulated, becomes a powerful oncogene found in many human cancers, including DLBCL.  DLBCL is the most common form of non-Hodgkin’s lymphoma and has two major subtypes: germinal center B-cell-like (GCB) and activated B-cell-like (ABC).  When compared to the GCB form, ABC lymphomas respond much more poorly to current therapies and often exhibit overexpression or overactivation of STAT3. To investigate how STAT3 might contribute to this aggressive phenotype, we have used ChIP-Seq to identify STAT3 binding sites in 8 DLBCL cell lines (4 GCB subtype, 4 ABC) that are derived from patient tumors.  10,337 distinct STAT3 binding regions are occupied in at least two of the eight cell lines. One third (n = 3524) are differentially bound by STAT3 between the two subtypes (FDR < 0.05).  More BRs are strongly bound in ABC than in GCB: 44% of differentially bound BRs (n = 1550) show more STAT3 binding in GCB, while 56% (n = 1974) are more strongly bound in ABC.

overall_design: Identification and comparison of STAT3 transcription factor binding sites in 8 cell lines that represent the 2 subtypes of DLBCL.  4 cell lines are subtyped as ABC and 4 are subtyped as GCB.  2-9 replicates and 1 input control are present for each cell line.  (Cell line OCI-Ly19 was not included in the final analysis because RNA-Seq showed that its gene expression clustered in between the subtypes, probably due to its EBV+ status.  However, its peak calls were used in intermediate steps of the analysis pipeline.  Its sequencing runs have been included for completeness.)

last: Ouyang

last: Zhang

last: Kundaje

last: Lacroute

last: Snyder


title: Regulation of Trypanosoma brucei total and polysomal mRNA during development within its mammalian host

last: Ivens

summary: The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically ‘slender forms’ to transmissible ‘stumpy forms’ through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched in throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms), irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been identified to enrich transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream haves been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.

overall_design: Examination of gene expression during life cycle stages.

last: Matthews


title: Determination of the spatial organization of the FXN gene locus by using Chromosome Conformation Capture technique coupled with High-throughtput sequencing (3C-seq).

first: Sanjay

middle: Padmakar

last: Khadayate

address: Du Cane Road

institute: MRC London Institute of Medical Sciences

summary: Accumulating data from different groups have demonstrated that alteration of post-translational histone modifications is an important underlying mechanism for FXN silencing in FRDA. The relationship between chromatin architecture and histone modification marks in the FXN gene locus is likely to be important for understanding the silencing mechanism. We therefore investigated the spatial organization of the FXN gene locus using the 3C-sequencing method.

overall_design: EBV transformed lymphoblastoid cell lines derived from patients (GM15850, GM16234 and GM16798) and Healthy control (GM14664 and GM15851) were used for generating 3C-seq libraries.

first: Ping

last: Chan


title: H3K4 mono- and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation

first: Kai

last: Ge

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

overall_design: Refer to individual Series


title: Correlation between STAT3 binding presence and gene expression levels in subtypes of diffuse large B cell lymphoma


title: Cohesin and CTCF Differentially Affect the Chromatin Architecture and Gene Expression in Human Cells

city: La Jolla

first: Jesse

last: Dixon

address: 10010 N. Torrey Pines Rd.

institute: Salk Institute for Biological Studies

summary: Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many self-associating topological domains. The boundary sequences are enriched for binding sites of CTCF and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains.  To determine the role of cohesin and CTCF in higher order chromatin architecture in human cells, we proteolytically cleaved the cohesin complex from interphase chromatin and examined changes in chromosomal organization as well as transcriptome. We observed a general loss of local chromosomal interactions upon disruption of cohesin complex, but the topological domains remain intact. However, we found that depletion of CTCF by RNA interference in these cells not only reduced intra-domain interactions but also increased inter-domain interactions. Further more, distinct groups of genes become mis-regulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute in different ways to chromatin organization and gene regulation.

overall_design: Hi-C and mRNA-seq experiments in Cohesin and CTCF depleted HEK293 cells

first: Bing

last: Ren

first: Kerstin

last: Wendt


title: Constitutive activation of the receptor-like kinase NIK impairs translation and confers tolerance to begomovirus infection

city: Viçosa

first: Elizabeth

middle: Pacheco Batista

last: Fontes

state: MG

address: Av. PH. Rolfs, s/n

department: Biochemistry

country: Brazil

institute: Federal University of Viçosa

summary: To generate an efficient defense against begomovirus, we modulated the activity of the immune defense receptor NIK (NSP-Interacting Kinase) in tomato plants; NIK is a virulence target of the begomovirus NSP during infection. Replacing threonine-474 with aspartate (T474D) within the kinase activation loop promoted the constitutive activation of NIK-mediated defenses. This activation resulted in the down-regulation of translation-related genes and the suppression of global translation in T474D-overexpressing tomato lines. We also found that T474D-induced defense-related transcripts were associated with polysomes and immune proteins, which accumulated to detectable levels in T474D leaves. Consistent with these findings, T474D transgenic lines were tolerant to the tomato-infecting begomoviruses ToYSV and ToSRV. We propose that NIK mediates an anti-viral response via translation suppression and immune system induction.

overall_design:
Global variation on gene expression induced by NIK  expression and virus infection using total RNA from mock-inoculated and ToYSV-infected tomato wild-type plants,  mock-inoculated and infected 35S::NIK1-4 overexpressing lines and mock-inoculated and infected 35S::T474D overexpressing lines.
File map_itag23.csv correlates the ITAG 2.3 cDNA ID with the 21 bp reads in file Profiles_with_differential_expressions.csv.

first: Otavio

last: Brustolini

first: Anesia

last: Santos


title: Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis

status: Public on Dec 15 2013

city: Dallas

first: Hume

last: Stroud

state: TX

address: 5323 Harry Hines Blvd

institute: UT Southwestern

summary: DNA methylation occurs in both CG and non-CG sequence contexts. Non-CG methylation is abundant in plants, and is mediated by CHROMOMETHYLASE (CMT) and DOMAINS REARRANGED METHYLTRANSFERASE (DRM) proteins; however its roles remain poorly understood. Here we characterize the roles of non-CG methylation in Arabidopsis thaliana. We show that a poorly characterized methyltransferase, CMT2, is a functional methyltransferase in vitro and in vivo. CMT2 specifically binds histone H3 lysine 9 (H3K9) dimethylation and methylates non-CG cytosines at sites that are also regulated by H3K9 dimethylation. By generating different combinations of non-CG methylation mutants, we reveal the contributions and redundancies between each methyltransferase in DNA methylation patterning and in regulating transposable elements (TEs) and protein-coding genes. We also demonstrate extensive dependencies of small RNA accumulation and H3K9 methylation patterning on non-CG methylation, suggesting self-reinforcing mechanisms between these epigenetic factors. The results suggest that non-CG methylation patterns are critical in shaping the histone modification and small non-coding RNA landscapes.

overall_design: Eighteen mRNA-seq samples, five smRNA-seq samples, five bisulfite-seq samples, twenty ChIP-seq samples. Bisulfite-seq data for cmt2-7 single mutants, cmt3 single mutants, drm1/2 double mutants, drm1/2 cmt3 triple mutants are deposited in GSE39901. Processed wiggle format files for all datasets can be downloaded at http://genomes.mcdb.ucla.edu/AthBSseq/


title: Total transcriptome profiling of the Atclb1-1 loss of function mutant and wild type of Arabidopsis

city: Little Rock

first: Mariya

last: Khodakovskaya

state: AR

address: 2801 S. University Avenue

department: Biology

institute: University of Arkansas at Little Rock

summary: Since AtCLB act as a negative regulator of abiotic stress response, we asked the question as to what additional factors might be contributing towards stress tolerance in Atclb1-1 loss of function mutants. One approach to find answers to this question would be to see which pathways or genes are being affected in mutant plants due to the disruption of AtCLB gene function. To achieve this, we sought a global transcriptome profiling of the mutant plant in comparison to its wild type counterpart.

overall_design: We analyzed the Atclb1-1 loss of function mutant and wild type Arabidopsis transcriptomes by RNA sequencing in an Illumina genome analyzer platform. From the analysis, we grouped all differentially expressed genes into various functional categories based on their biological process, molecular function and cellular component

first: Aleksandra

last: Markovets


title: Analysis of transgene siRNAs and ARL-8-dependent siRNAs in Caenorhabditis elegans

first: Sylvia

middle: E. J.

last: Fischer

address: 185 Cambridge Street, CPZN-7250

department: Molecular Biology

institute: Massachusetts General Hospital

summary: We analyzed the C. elegans small RNA response to high copy transgene sequences expressed in the soma in a wild type and an eri-6/7 mutant background. We also analyzed small RNA defects in the arl-8(tm2472) mutant. Transgene siRNAs are 22 nt long, mostly antisense, and correspond to the promoter, coding regions, the 3'UTR and plamsid sequences present on the transgene.  Transgene siRNAs are decreased in the eri-6/7 mutant. In the arl-8 mutant, 26G siRNAs in the ALG-3/4 dependent endogenous RNAi pathway are decreased.

overall_design: Sequencing small RNAs from C. elegans transgenic strains and mutants.

middle: E

first: Gary

last: Ruvkun


title: L-Myc expression by dendritic cells is required for optimal T-cell priming

status: Public on Dec 14 2013

city: Saint Louis

first: Kenneth

last: Murphy

state: MO

address: 660 S. Euclid Ave.

department: Pathology and Immunology

institute: Washington University School of Medicine

summary: The transcription factor IRF8 is a critical regulator of plasmacytoid dendritic cell (pDC) and classical dendritic cell (cDC) development in both mouse and man.  Yet the downstream molecular targets that regulate DC homeostasis and development are largely unknown.  A recent study using gene expression analysis of IRF8-deficient myeloid and lymphoid progenitors identified the Myc paralog Mycl1 as a potential transcriptional target of IRF8.  We report here that Mycl1 is a mediator of DC homeostasis at steady state and during inflammation, and its expression is regulated by IRF8 in multiple DC lineages.  We have further validated these observations with ChIP-Seq of IRF8 binding to the Mycl1 locus.  Notably, IRF8 binding to Mycl1 locus is independent of an interaction with the AP1 factor, BATF3.  Additionally, our genome-wide survey of IRF8 binding identified both EICE and AICE motifs.

overall_design: Examination of IRF8 binding in dendritic cells

first: Wumesh

last: KC


title: Mapping and dynamics of regulatory DNA in A. thaliana accessions

city: SEATTLE

first: John

middle: A

last: Stamatoyannopoulos

state: WA

address: 2211 Elliott Avenue, 6th Floor

department: Genome Sciences

institute: Altius Institute / University of Washington

summary: We performed DNase-seq on 7-day-old seedlings from four A. thaliana accessions: Bur-0, Tsu-0, Bay-0, Est-1 using INTACT constructs (Deal and Henikoff, Developmental Cell, 2010) driven by the UBQ10 promoter.

overall_design: Chromatin accessibility profiling (Dnase I-seq) of 7-day-old seedlings of A. thaliana accessions: Bur-0, Tsu-0, Bay-0 & Est-1 grown in LD conditions (16hr light 22°C, 8hr dark 20°C).

first: James

last: Urton

first: Alessandra

last: Sullivan

first: Kerry

last: Bubb

first: Andrej

last: Arsovski

first: Peter

last: Sabo

first: Richard

last: Sandstrom

first: Robert

last: Thurman

first: Shane

last: Neph

first: Alex

last: Reynolds

first: Audra

middle: K

last: Johnson

first: Eric

last: Haugen

first: Agnieszka

last: Dargiel

first: Jun

last: Neri

first: Molly

last: Weaver

first: Morgan

last: Diegel

first: Jennifer

last: Nemhauser

first: Christine

last: Queitsch


title: Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo

city: Cambridge

first: Silvi

last: Rouskin

address: 455 Main Street

institute: Whitehead Institute

summary:
We present an approach for globally monitoring RNA structure in native conditions in vivo with single nucleotide precision. This method is based on in vivo modification with dimethyl sulfate (DMS), which reacts with unpaired adenine and cytosine residues9, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known mRNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome10. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast reveals that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding, thermodynamics play an incomplete role in determining mRNA structure in vivo.
We use Dimethyl Sulfate to probe the structure of rRNA and mRNA in yeast in vivo, in vitro, and at different temperatures in vitro. We obtain a great agreement between in vivo data and known mRNA structures as well as the ribosome crystal structure. We find that in contrast  to ribosomal rna, mRNAs are less structured in vivo than in vitro, and the structures present in vivo can only partially be explained by thermodynamic stability. In addition, we identify new regulatory structures present in vivo.

sample_organism:
Saccharomyces cerevisiae
Homo sapiens

platform_organism:
Saccharomyces cerevisiae
Homo sapiens

overall_design:
Examination of RNA structure in yeast under different conditions - in vivo and in vitro at five different temperatures (30,45,60,75,95)
We adapt our DMS-seq assay for use in mammalian cells and probe RNA structure genome-wide in K562 cells.
We probe the RNA structure of primary fibroblast using DMS on a genome-wide scale to confirm the presence of more structures in vitro. In addition we probe the RNA structure in yeast upon ATP depleted conditions to investigate whether active (ATP-dependent) processed are modulating RNA structure in vivo.

first: Meghan

last: Zubradt

first: Stafan

last: Washietl

first: Manollis

last: Kellis

first: Jonathn

middle: S

last: Weissman


title: High-resolution mapping of transcription factor binding sites on native chromatin

city: Seattle

first: Jorja

last: Henikoff

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary: Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites.

sample_organism:
Saccharomyces cerevisiae
Drosophila melanogaster

overall_design: Chromatin immunoprecipitation of micrococcal nuclease-digested native chromatin followed by paired-end sequencing (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin 'ORGANIC' profiling) of DNA-binding proteins Abf1 and Reb1 from S. cerevisiae and GAGA-binding factor (GAF) and Pipsqueak (Psq) from D. melanogaster S2 cells; and, Sono-seq (paired-end sequencing of formaldehyde cross-linked and sonicated chromatin) of yeast nuclei. Reb1 ORGANIC profiling was performed at three different salt (NaCl) concentrations (80, 150, and 600 mM) and Abf1 ORGANIC profiling was done at two different salt concentrations (80 and 600 mM) to achieve varying levels of stringency. GAF and Psq ORGANIC profiles were determined at 80 mM salt. Two replicates each of Reb1 and Abf1 600 mM ORGANIC experiments, mixed Drosophila S2 cell and S. cerevisiae nuclei Reb1 ORGANIC experiments, yeast Sono-seq, and GAF and Psq ORGANIC experiments were performed. Each S. cerevisiae and mixed S2 cell/yeast ORGANIC profiling experiment included separately sequenced input chromatin and ChIP samples. Total of 24 samples.

first: Sivakanthan

last: Kasinathan

first: Guillermo

last: Orsi

first: Gabriel

last: Zentner

first: Kami

last: Ahmad

first: Steven


title: Commonality in small RNA profiles between transgene-induced and naturally occurring RNA silencing of chalcone synthase-A gene in petunia

city: Ueda

first: Hideo

last: Matsumura

state: Nagano

address: Tokita 3-15-1

department: Gene Research Center

institute: Shinshu University

summary:
Background: Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs.
Results: We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved.
Conclusions: The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the biosynthetic processes of siRNAs including cleavage of CHS-A transcripts and subsequent production of secondary siRNAs in exon 2. The data also suggest that these events occurred at multiple sites, which can be a feature of these silencing phenomena.

overall_design: Small RNAs isolated from petal tissues in two petunia lines were analyzed.

first: Megumi

last: Kasai

first: Kentaro

last: Yoshida

first: Ryohei

last: Terauchi

first: Akito

last: Taneda

first: Akira

last: Kanazawa


title: Deep sequencing reveals divergent expression patterns within the small RNA transcriptomes of cultured and vegetative tissues of sugarcane

status: Public on Dec 13 2013

city: Brisbane

first: Peter

middle: Richard

last: Sternes

state: Queensland

address: John Hines Building, University of Queensland, St Lucia

department: School of Agriculture and Food Science

country: Australia

institute: University of Queensland

summary: We report the deep sequencing of small RNA populations derived from apex, internode 3, internode 6, internode 16, leaf +3, calli and suspesion cells from sugarcane cultivar Q117

overall_design: Each vegetative tissue was sampled from three independent field grown plants, pooled and sequenced using the GAIIx platform. Calli and suspension cell cultures were sequenced using the HiSeq 2000 platform.

middle: R

first: Richard

last: Moyle


title: Differences in the global transcriptome between high- and low-pathogenic Salmonella Enteritidis strains

city: Pullman

first: Devendra

last: Shah

address: #402, Bustad Hall

department: Veterinary Microbiology and Pathology

institute: Washington State University

summary: We report the use of RNA-Seq technique to characterize differences in the global transcritome between multiple genetically similar but phenotypically diverse poultry-associated field strains of S. Enteritidis grown in laboratory media at avian body temperature (42°C).

sample_organism: Salmonella enterica subsp. enterica serovar Enteritidis

overall_design: Characterization and comparison of transcriptome of six S. Enteritidis strains previously identified as high-pathogenic (HP, n=3) and low-pathogenic (LP, n=3)


title: Targeting transcriptional dependencies in cancer using a covalent CDK7 inhibitor (ChIP-Seq)

status: Public on Dec 23 2013

last: Young

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: Cyclin-dependent kinase 7 (CDK7) plays a critical role in the general regulation of RNA polymerase II-mediated transcription.  However, the absence of selective CDK7 inhibitors has hindered the ability to investigate the consequences of acute and prolonged inhibition of CDK7 under normal and pathological conditions.  Here we present the discovery and characterization of the first covalent CDK7 inhibitor, CDK7-IN-1, that has the unprecedented ability to target a unique cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7 amongst the 20 known CDKs.  Cancer cell line profiling indicates that a subset of cancer cell lines, including T-cell acute lymphoblastic leukemia (T-ALL), exhibit 100-fold greater sensitivity to CDK7-IN-1 over other tumor and normal cell lines.  Genome-wide expression analysis in Jurkat T-ALL indicates that CDK7-IN-1 disproportionally affects RUNX1 as well as other components of the TAL1 transcriptional network and its targets, downregulating key regulators of transcription and apoptosis critical for the T-ALL state.   These oncogenes are encoded by short-lived mRNA transcripts, are associated with super-enhancers, and exhibit a strong dependency on continuous transcription for sustained expression.  Therefore, pharmacological modulation of CDK7 kinase activity may define a method for the identification and treatment of tumor types exhibiting extreme dependencies on transcription for maintenance of the oncogenic state.

overall_design: Jurkat, MM1S, Loucy, and HeLa (WT and Dox-inducible CDK7 mutant) cells were treated with various drugs including a covalent inhibitor of CDK7 (CDK7-IN-1), a reversible inhibitor of CDK7 (CDK7-IN-1), Flavopiridol, Actinomycin D, and DMSO controls.  Replicates are annotated.

first: Nicholas

middle: P

last: Kwiatkowski

first: Tinghu

last: Zhang

middle: B

last: Rahl

first: Brian

middle: J

last: Abraham

first: Jessica

last: Reddy

first: Scott

last: Ficarro

first: Anahita

last: Dastur

first: Sridhar

last: Ramaswamy

first: Arnaud

last: Amzallag

first: Bethany

last: Tesar

first: Christopher

last: Jenkins

first: Nancy

middle: M

last: Hannett

first: Douglas

last: McMillin

first: Takaomi

last: Sanda

first: Taebo

last: Sim

first: Nam

middle: D

last: Kim

first: Thomas

last: Look

first: Constantine

last: Mitsiades

first: Andrew

last: Weng

first: Jennifer

last: Brown

first: Cyril

last: Benes

first: Jarrod

last: Marto

first: Nathanael

last: Gray


title: ChIP-Seq of transcriptional regulators in MM1S cells upon small molecule treatment.

first: Richard

last: Young

summary: Discovery of the genome-wide location of proteins using ChIP-Seq has allowed global mapping of the key transcription factors and chromatin regulators that control gene expression programs in various cells. Many DNA-associated processes are targeted for disease therapy. This study investigates the functions of small molecule therapeutics that target DNA-associated processes involved of CDK9 and BRD4.

overall_design: Genomic DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. ChIP was performed using an antibody against RNAP2, BRD2, BRD3, BRD4, CDK8, CDK9, H3K27me3, H3K20me3, and CTCF using whole cell extract (WCE) as a background control.

first: Anders

last: Lars

first: Heather

last: Hoke

first: Rahl

last: Peter


title: Chem-Seq of chemical derivatives in MM1S cells

summary: An ability to map the global interactions of a chemical entity with chromatin genome-wide could provide new insights into the mechanisms by which a small molecule perturbs cellular functions. we developed a method that uses chemical derivatives and massively parallel DNA sequencing (Chem-Seq) to identify the sites bound by small chemical molecules throughout the human genome.

overall_design: We developed in vivo and in vitro Chem-Seq protocols with a biotinylated derivative of small molecules.  In the in vivo protocol, Cells were first treated with biotinylated ligand and cross-linked with formaldehyde at the same time.  Cells were then lysed, sonicated to shear the DNA, and streptavidin beads were used to isolate biotinylated ligand and associated chromatin fragments.  We then used massively parallel sequencing to identify the enriched DNA fragments, and mapped these sequences to the genome. In in vitrol protocol, MM1.S cells were fixed and the derived sonicated lysate incubated with biotinylated drug to enrich for bound chromatin regions in vitro. We then used massively parallel sequencing to identify the enriched DNA fragments, and mapped these sequences to the genome.

first: Matthew

middle: G

last: Guenther


title: Twist expression in HMLE and breast cancer T47D cells

status: Public on Dec 12 2013

city: Lexington

first: Binhua

last: Zhou

state: KY

address: 789 South Limestone

department: Markey Cancer Center/Biochemistry

institute: University of Kentucky School of Medicine

summary:
Twist is a key EMT inducer, expression of Twist will induce EMT in HMLE and breast tumor T47D cells
By expressing Twist in HMLE and T47D cells, which lack the expression of Twist, will identify the genes regulated by Twist

overall_design: Expressing Twist in HMLE and T47D cells, stable clones were selected and treated with BET inhibitor JQ1 and RNA were prepared for microarray analysis

first: Jian

last: Shi


title: The relationship between DNA methylation, genetic and expression inter-individual variation in untransformed human fibroblasts

city: Montreal

first: Stephan

last: Busche

state: Quebec

address: 740 Dr. Penfield Avenue

department: Human Genetics

institute: McGill University

type: Methylation profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Coordinated genomic control of ciliogenesis and cell movement by Rfx2

city: Ulsan

first: Taejoon

last: Kwon

address: 50 Unist-gil

department: Department of Biomedical Engineering

country: South Korea

institute: Ulsan National Institute of Science and Technology

summary: We have performed a systems-level analysis of the RFX/Daf-19 family transcription factor, Rfx2. Using a combination of high-throughput sequencing of Rfx2-regulated transcripts and chromosomal binding sites, we provide a comprehensive accounting of the target genes by which Rfx2 controls ciliogenesis and cilia beating in vertebrates.

overall_design: RNA-seq: two biological replicates for control and RFX2 knockdown by morpholino injection, ChIP-seq: RFX2-GFP pulldown with GFP antibody, GFP only expression used as control

first: Mei-I

last: Chung

first: Rakhi

last: Gupta

first: Julie

middle: C

last: Baker

first: Edward

last: Marcotte

first: John

last: Wallingford