title: Tracking the fate of pathogenic CD4 T helper cells in vivo.

status: Public on Apr 25 2015

city: Honolulu

first: Travers

last: Ching

state: HI

address: 701 Ilalo St #600

department: Epidemiology

institute: University of Hawaii Cancer Center

summary: Inflammation is a beneficial host response to infection, but it also contributes to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases.  These cells exhibit both instability (i.e., they can cease to express their signature cytokine, IL-17A) and plasticity (i.e., they can start expressing cytokines typical of other lineages) upon in vitro re-stimulation. However technical limitations prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in  expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as “transdifferentiation”. Furthermore, while Th17 cell instability/plasticity has been associated with pathogenicity, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate.  Here we used two novel fate-mapping mouse models to track Th17 cells during immune responses to show that CD4+ T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a global change in their transcriptome and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF- β signaling and the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases.

overall_design: We isolated intestinal lymphocytes from two independent experiments, each using 5 mice injected with anti-CD3 mAb. Th17, exTh17, Tr1 exTh17, Tr1, Foxp3 Treg and Foxp3 IL-10+ Treg cell populations were FACS-sorted from these two independent experiments and the cells of each population were pooled before the analysis. Around 5,000 cells for each population were processed.

first: Nicola

last: Gagliani

first: Andrea

last: Iseppon

first: Carolina

last: Amezcua Vesely

first: Leonie

last: Brockmann

first: Noah

middle: W

last: Palm

first: Marcel

middle: R

last: de Zoete

first: Paula

last: Licona-Limon

first: Ricardo

last: Paiva

first: Zi

last: Xiaoyuan

first: Xinghua

last: Pan

first: Rong

last: Fan

first: Lana

last: Garmier

first: Jens

last: Geginat

first: Brigita

last: Stockinger

first: Enric

last: Esplugues

first: Samuel

last: Huber

first: Richard

last: Flavell


title: Suppressed 60S ribosome exportation in maize ribosome export associated 1 mutant triggers inconsistent transcriptional and translational regulations

city: Shanghai

first: Weiwei

last: Qi

address: 333 Nanchen Road

country: China

institute: Shanghai University

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Transcriptome analysis of Dunaliella viridis in response to changes in light duration and temperature

status: Public on Apr 27 2015

city: Raleigh

first: Brian

last: Howard

state: NC

address: 1428 Sandusky Lane

department: Plant Biology

country: USA

institute: North Carolina State University

overall_design: Time course experiment  experiment with 5 evenly spaced time points, t1-t5, and two experimental factors: a) light condition (2 levels: continuous light and light/dark cycle) and b) temperature (2 levels: 25 degrees Celsius and 35 degrees Celsius). Cells grown in the light/dark condition were exposed to a 12 hour light/dark cycle, whereas continuous light samples were grown under constant light conditions.  All cells were grown at 25 degrees Celsius for timepoints t1 and t2; at time t3, cells grown in the high temperature condition were raised to 35°C, and kept at this temperature for the remainder of the experiment.  Bar-coded samples were sequenced in two separate sequencing runs using 100-nt Illumina RNA-Seq reads. In the first run a single pooled biological replicate was sequenced from each of the 12 combinations of time, light condition and temperature from time points t3 to t5. The bar-coded samples were divided among 4 Illumina Lanes resulting in 48 distinct sequencing data files. In the second run ("Run 2") a single bar-coded biological replicate was sequenced from each of the 10 combinations of time and light condition from time points t1 through t5, with temperature fixed at 25°C.

first: Heike

last: Sederoff


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and rea1 mutant Transcriptomes

summary: Analysis of  gene expression level. The hypothesis tested in the present study was that rea1 mutant influence the expression of translation and nucleosome assembly associated genes. Results provide important information of the gene expression level regulation of ribosome proteins, elongation factors, histones and genes associated with other biological processes.

overall_design: Endosperm mRNA profiles of 15DAP wild type (WT) and rea1 mutant were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500 (Zea mays).

first: Jie

last: Zhu


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and rea1 mutant total and polysome-bound Transcriptomes

summary: Analysis of  gene expression level. The hypothesis tested in the present study was that rea1 mutant influence the translation level of translation and nucleosome assembly associated genes. Results provide important information of the gene translation level regulation of ribosome proteins, elongation factors, histones and genes associated with other biological processes.

overall_design: Endosperm total and polysome-bound mRNA profiles of 15DAP wild type (WT) and rea1 mutant were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500 (Zea mays).


title: Pharmacological HDAC inhibition attenuates cardiac hypertrophy and histone acetylation of target genes

city: Melbourne

first: Assam

last: El-Osta

address: 75 Commercial Road

country: Australia

institute: Baker Heart and Diabetes Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Cardiac hypertrophy is characterized by an increase in heart size and profound gene expression changes. Pharmacological histone deacetylase (HDAC) inhibitors attenuate pathological cardiac remodeling and hypertrophic gene expression. Published literature has linked enzymes that mediates histone acetylation to pathogenesis, however, the role of histone acetylation to define hypertrophic gene regulatory events are not well understood. We used chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (seq) to comprehensively examine genome-wide histone acetylation changes in a pre-clinical model of pathological cardiac hypertrophy by transverse aortic constricted (TAC). We examined the gene targets conferred by the prototypical HDAC inhibitor Trichostatin A (TSA). TSA induces genome-wide histone acetylation and deacetylation in the heart. Alterations to histone acetylation were found on genes involved in cardiac morphology such as cardiac contraction, collagen deposition, inflammation and extracellular matrix. Gene set enrichment analysis identified NF-kappa B (NFKB), as a transcription factor positively correlated with TAC and negatively correlated with TSA by ChIP-seq. Histone acetylation on the promoters of NKFB target genes was increased, consistent with gene activation. In contrast, the promoters of these genes were deacetylated by TSA in hypertrophic animals and reduced expression of NFKB target genes. Our results suggest a potential mechanism for TSA mediated cardioprotection conferred by histone deacetylation of target genes implicating the importance of inflammation.

overall_design: ChIP-seq profiles for histone acetylation in left ventricles of mice that develop cardiac hypertrophy and treated with HDAC inhibitors were generated by deep sequencing, using Illumina GAIIx.

first: Jenny

last: Ooi

first: Natasha

last: Tuano

first: Haloom

last: Rafehi

first: Xiao-Ming

last: Gao

first: Mark

last: Ziemann

first: Xiao-Jun

last: Du


title: Growth signals employ CGGBP1 to suppress transcription of Alu-SINEs [ChIP-Seq]

status: Public on Apr 29 2015

city: Gandhinagar

first: Umashankar

last: Singh

state: Gujarat

address: Acad Block 4 Room 317 IIT Gandhinagar Palaj

department: Biological Sciences and Engineering

country: India

institute: IIT Gandhinagar

summary: Alu SINEs are the most numerous frequently occurring transcription units in our genomes and possess sequence competence for transcription by RNA Pol III. However, through poorly understood mechanisms, the Alu RNA levels are maintained at very low levels in normal somatic cells with obvious benefits of low rates of Alu retrotransposition and energy-economical deployment of RNA Pol III to the tRNA genes which share promoter structure and polymerase requirements with Alu SINEs. Using comparative ChIP sequencing, we unveil that a repeat binding protein, CGGBP1, binds to the transcriptional regulatory regions of Alu SINEs thereby impeding Alu transcription by inhibiting RNA Pol III recruitment. We show that this Alu-silencing depends on growth factor stimulation of cells and subsequent tyrosine phosphorylation of CGGBP1. Importantly, CGGBP1 ensures a sequence-specific discriminative inhibition of RNA Pol III activity at Alu promoters, while sparing the structurally similar tRNA promoters. Our data suggest that CGGBP1 contributes to growth-related transcription by preventing the hijacking of RNA Pol III by Alu SINEs.

overall_design: Examination of one DNA binding protein in two different conditions of treatment.

first: Prasoon

last: Agarwal

first: Bengt

last: Westermark


title: Small molecule inhibition of ERK dimerization prevents tumorigenesis by Ras-ERK pathway oncogenes

status: Public on Apr 24 2015

city: SANTANDER

first: Piero

last: Crespo

state: Cantabria

address: c/Albert Einstein, 22

country: Spain

institute: IBBTEC

summary: About 50% of human malignancies exhibit unregulated signalling through the Ras-ERK1/2 (ERK) pathway, as a consequence of activating mutations in members of Ras and Raf families. However, the quest for alternative Ras-ERK pathway-directed therapies is desirable. Upon phosphorylation ERK dimerize. We had previously demonstrated that dimerization is essential for ERK extranuclear but not nuclear signaling. Furthermore, by molecular biology approaches, we showed that specifically inhibiting ERK extranuclear component, by impeding ERK dimerization, is sufficient for curtailing tumor progression. Here, we have identified a small molecule inhibitor for ERK dimerization in vitro and in vivo that, without affecting ERK phosphorylation, prevents tumorigenesis driven by Ras-ERK pathway oncogenes, both in cellular and animal models. Importantly, this compound is unaffected by resistance-acquisition processes that hamper “classical” Ras-ERK pathway inhibitors. Thus, ERK dimerization inhibitors provide the proof of principle for two novel concepts in cancer therapy: 1) The blockade of sublocalization-specific sub-signals, rather than total signals, as a means of effectively counteracting oncogenic Ras-ERK signaling. 2) Targeting regulatory protein-protein interactions such as dimerization, rather than catalytic activities, within a signaling route, as an approach for producing effective anti-tumoral agents. Strategies aimed at preventing aberrant flux through this route remain an attractive option for therapeutic intervention in cancer. In this respect, drugs inhibiting the kinase activities of BRaf and MEK have yielded promising results.

overall_design: A375p cells treated with10 μM of either DEL22379, SCH772984 or DMSO as a control for two hours. mRNA from A375p cells was extrated using RNeasy mini kit (Qiagen, Germany) according to the manufacturer's instructions. Cells were previously treated with10 μM of either DEL22379, SCH772984 or DMSO as a control for two hours.


title: Genome-wide assessments of transcriptional pauses and errors by nascent transcript sequencing combined with RNase footprinting (RNET-seq)

city: Frederick

first: Masahiko

last: Imashimizu

state: MD

department: NCI/CCR

institute: NCI

summary:
We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli.

We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage.

overall_design: The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.


title: KLF4 Dependent Phenotypic Modulation of SMCs Plays a Key Role in Regulating Plaque Pathogenesis

city: Charlottesville

first: Gary

middle: K

last: Owens

state: VA

address: 415 Lane Road MR5 Building Room 1322

institute: University of Virginia

summary: Cardiovascular disease is the leading cause of death in developed countries and there is compelling evidence that the majority of these fatalities are secondary to rupture of unstable atherosclerotic plaques. However, the mechanisms that control plaque stability are poorly understood, although it is widely believed that plaques having a decreased ratio of cells positive for smooth muscle cell (SMC) markers such as ACTA2 relative to macrophage markers are more likely to rupture. Herein we employMyh11-CreERT2 ROSA floxed STOP eYFP Apoe-/- mice to trace SMC lineage and show that traditional methods for detecting SMCs based on immunostaining for SMC markers like ACTA2 are unable to detect 82% of SMC-derived cells within advanced atherosclerotic lesions. Moreover, we show that these SMC-derived cells within advanced lesions exhibit multiple phenotypes including activation of multiple markers of macrophages, mesenchymal stem cells (MSC), and myofibroblasts (MF). Importantly, we show that SMC-derived macrophage like cells are phagocytic based on electron microscope YFP immunolabeling. In addition, results of single cell epigenetic assays developed in our lab shows that nearly 20% of macrophage-like cells within human lesions are of SMC not myeloid origin. These phenotypic transitions appear to be critical in lesion pathogenesis, in that we show that SMC-specific conditional knockout (KO) of the pluripotency factor, Krüppel-like factor 4 (KLF4), resulted in marked reductions in lesion size, increases in multiple indices of plaque stability, and reduced numbers of SMC-derived MSC- and macrophage-like cells. Finally, we show that cholesterol loading of cultured SMC is associated with activation of multiple markers of macrophages and MSC, secretion of pro-inflammatory cytokines, and increased phagocytosis, all of which are Klf4 dependent. Taken together, results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that loss of Klf4 within SMC result in major changes in SMC phenotype and function that play a major role in lesion pathogenesis.

overall_design: Examination of KLF4 transcription factor in an atherosclerosis setting

first: Laura

middle: S

last: Shankman

first: Delphine

last: Gomez

first: Morgan

last: Salmon

first: Olga

middle: A

last: Cherepanova

first: Gabriel

middle: F

last: Alencar

first: Pamela

last: Swiatlowska

first: Ryan

middle: M

last: Haskins

first: Alexandra

last: Newman

first: Elizabeth

last: Greene

first: Adam

last: Straub

first: Brant

last: Isakson

first: Gwendalyn

middle: J

last: Randolph


title: Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed

city: Berlin

first: Petar

last: Glazar

address: Robert Rössle Str. 10 (H. 87)

department: Systems Biology of Gene Regulatory Elements

country: Germany

institute: Max Delbrück Center for Molecular Medicine

summary: Circular RNAs (circRNAs) are an endogenous class of animal RNAs. Despite their abundance, their function and expression in the nervous system are unknown. Therefore, we sequenced RNA from different brain regions, primary neurons, isolated synapses, as well as during neuronal differentiation. Using these and other available data, we discovered and analyzed thousands of neuronal human and mouse circRNAs. circRNAs were extraordinarily enriched in the mammalian brain, well conserved in sequence, often expressed as circRNAs in both human and mouse, and sometimes even detected in Drosophila brains. circRNAs were overall upregulated during neuronal differentiation, highly enriched in synapses, and often differentially expressed compared to their mRNA isoforms. circRNA expression correlated negatively with expression of the RNA-editing enzyme ADAR1. Knockdown of ADAR1 induced elevated circRNA expression. Together, we provide a circRNA brain expression atlas and evidence for important circRNA functions and values as biomarkers.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: To assess circRNA expression in mammalian brain, we sequenced and analyzed mouse brain regions (hippocampus, cerebellum, prefrontal cortex and olfactory bulb), various neuronal differentiation (mouse P19 and human SH-SY5Y cells) and maturation (mouse cortical neurons) stages, and subcellular compartments in mouse (synaptoneurosomal fraction, cytoplasmic fraction, whole brain lysate).

first: Agnieszka

last: Rybak-Wolf

first: Christin

last: Stottmeister

first: Natalia

last: Pino

first: Sebastian

last: Giusti

first: Mor

last: Hanan

first: Mikaela

last: Behm

first: Osnat

last: Bartok

first: Reut

last: Ashwal-Fluss

first: Marvin

last: Jens

first: Margareta

last: Herzog

first: Luisa

last: Schreyer

first: Panagiotis

last: Papavasileiou

first: Andranik

last: Ivanov

first: Marie

last: Öhman

first: Damian

last: Refojo

last: Kadener

first: Nikolaus

last: Rajewsky


title: Epigenetic and genetic features that lead to discovery of enhancer function

city: University Park

first: Ross

last: Hardison

state: PA

address: 303 Wartik Lab

institute: Pennsylvania State University

summary: The ability to measure epigenetic features, such as histone modifications and occupancy by transcription factors and co-activators, on a genome-wide scale is advancing the accuracy of CRM predictions. While integration of signals from multiple features is expected to improve predictions, the contribution of each feature to prediction accuracy is not known. We began with predictions of 4,915 erythroid enhancers based on genomic occupancy by TAL1, a key hematopoietic transcription factor that is strongly associated with gene induction in erythroid cells. Seventy of these DNA segments occupied by TAL1 (TAL1 OSs) were tested by transient transfections of cultured hematopoietic cells, and 56% of these were active as enhancers. Sixty-six TAL1 OSs were evaluated in transgenic mouse embryos, and 65% of these were active enhancers in various tissues. Inclusion of additional epigenetic features improved the prediction accuracy, with combinations of TAL1, GATA1, EP300, H3K4me1, and H3K27ac giving high accuracy of enhancer prediction (70%-75% success depending on method of clustering) while maintaining good sensitivity and specificity. Motifs that distinguish active from inactive TAL1 OSs implicate IRFs, STATs, and FOX protein families as candidate positive co-factors with TAL1, while REST (NRSF) and HOX family proteins are implicated in inactivity. While signals for evolutionary constraint were weak over the entire TAL1-bound DNA segments regardless of activity in either assay, phylogenetic preservation of a TF-binding site motif was associated with enhancer activity.

overall_design: The contribution of 8 epigenetic features including H3K27ac to identification of enhancers in 24h-induced G1E-ER4 cells.

first: Nergiz

last: Dogan

first: Weisheng

last: Wu

first: Christapher

last: Morrissy

first: Kuan-Bei

last: Chen

first: Aaron

last: Stronestrom

first: Maria

last: Long

first: Cheryl

last: Keller

first: Yong

last: Cheng

first: Deepti

last: Jain

first: Axel

last: Visel

first: Len

last: Pennacchio

first: Mitchell

last: Weiss

first: Gerd

last: Blobel

middle: C


title: Quality control of transcription start site selection by Nonsense-Mediated-mRNA Decay

city: Paris cedex 15

first: Alain

last: Jacquier

address: 25-28 rue du docteur roux

department: Génomes & génétique

country: France

institute: Institut Pasteur

summary: Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA quality-control pathway targeting transcripts such as messenger RNAs harboring premature stop-codons or short upstream open reading frame (uORFs). Our transcription start sites (TSSs) analysis of Saccharomyces cerevisiae cells deficient for RNA degradation pathways revealed that about half of the pervasive transcripts are degraded by NMD, which provides a fail-safe mechanism to remove spurious transcripts that escaped degradation in the nucleus. Moreover, we found that the low specificity of RNA polymerase II TSSs selection generates, for 47% of the expressed genes, NMD-sensitive transcript isoforms carrying uORFs or starting downstream of the ATG START codon. Despite the low abundance of this last category of isoforms, their presence seems to constrain genomic sequences, as suggested by the significant bias against in-frame ATGs specifically found at the beginning of the corresponding genes and reflected by a depletion of methionines in the N-terminus of the encoded proteins.

overall_design: 5'-end profile of WT and NMD deficient yeast cells

first: Frank

last: Feuerbach

first: Christophe

last: Malabat

first: Laurence

last: Ma

first: Cosmin

last: Saveanu


title: Transcript Profiling of Maize Embryo Sac and Preliminary Identification of Genes Involved in Embryo Sac-Pollen Tube Interactions

city: Tai An

first: shuaishuai

last: wang

address: daizong street

institute: ShanDong Agricultural University

summary: In this study, we identified the genes specifically/preferentially expressed in maize embryo sac and genes differentially expressed before and after pollination in maize embryo sac via RNA-seq. In total, more than 30% of them are unknown functions and more than 80% of which are first reported in maize embryo sac.

overall_design: Examination of 3 different tissues of maize to provide  a comprehensive and much more important data for further analysis of embryo sac-pollen tube interactions.

first: Xiansheng

last: Zhang


title: Onchocerca-derived small RNAs in host nodules and serum

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

country: United Kingdom

institute: Centre for Immunity, Infection and Evolution

type: Non-coding RNA profiling by high throughput sequencing

summary: We report the presence of Onchocerca ochengi and O. volvulus derived small RNAs in bovine nodule fluids and human serum and plasma, respectively. Further comparisons with other related filarial nematodes like Litomosoides sigmodontis and Dirofilaria immitis reveal common and distictive signatures associated to the Onchocerca species.

sample_organism:
Homo sapiens
Bos taurus

platform_organism:
Homo sapiens
Bos taurus

overall_design: Examination of small RNA content in bovine nodule fluids and human serum/plasma by Next Generation sequencing

first: Juan

last: Quintana

first: Benjamin

middle: L

last: Makepeace

first: Simon

last: Babayan

first: Kenneth

last: Pfarr

first: Mark

last: Blaxter

first: Alexander

last: Debrah

first: Samuel

last: Wanji

first: Henrietta

last: Ngangyung

first: Germanus

last: Bah

first: Vincent

middle: N

last: Tanya

first: David

last: Taylor

first: Achim

last: Hoerauf

first: Amy

middle: H

last: Buck


title: The histone methyltransferase Setd8 represses Gata2 expression and regulates erythroid maturation

city: Rochester

first: Laurie

last: Steiner

state: New York

address: 601 Elmwood Ave

department: Pediatrics

institute: University of Rochester

summary: The chromatin modifying enzymes that drive the erythroid-specific transcription program are incompletely understood. Setd8 is the sole histone methyltransferase in mammals capable of generating mono-methylated histone H4 lysine 20 (H4K20me1) and is expressed at significantly higher levels in erythroid cells than any other cell- or tissue- type, suggesting that Setd8 has an erythroid-specific function. To test this hypothesis, stable knockdown of Setd8 was established in extensively self-renewing erythroblasts (ESREs), a well-characterized, non-transformed, model of erythroid maturation. Setd8 knockdown impaired erythroid maturation, characterized by a delay in hemoglobin accumulation, larger cell area, persistent kit expression, incomplete nuclear condensation, and lower rates of enucleation than control cells. Setd8 knockdown did not alter ESRE proliferation or viability, or result in accumulation of DNA damage. Global gene expression analyses following Setd8 knockdown suggests that in erythroid cells, Setd8 functions primarily as a repressor and demonstrated high levels of Gata2 expression. Setd8 occupies critical regulatory elements in the Gata2 locus, and knockdown of Setd8 resulted in loss of H4K20me1 and gain of H4 acetylation at the Gata2 1S promoter. Taken together, these results imply that Setd8 is an important regulator of erythroid maturation that works in part through repression of Gata2.

overall_design: RNA-seq was performed of Setd8 knockdown and control cells, both while the cells were proliferating, and after 6 hours of maturation.


title: A library of native and synthetic yeast core promoters driving YFP expression

status: Public on Apr 23 2015

city: Rehovot

first: Eran

last: Segal

address: 234 Herzl St.

country: Israel

institute: Weizmann Institute

summary: The core promoter is the regulatory sequence to which RNA polymerase is recruited and where it acts to initiate transcription. Here, we present the first comprehensive study of yeast core promoters, providing massively parallel measurements of core promoter activity and of TSS locations and relative usage for thousands of native and designed sequences. We found core promoter activity to be highly correlated to the activity of the entire promoter, and that sequence variation in different core promoter regions substantially tunes its activity in a predictable way. We also show that location, orientation and flanking bases critically affect TATA element function, that transcription initiation in highly active core promoters is focused within a narrow region, that poly(dA:dT) orientation has functional consequence at the 3' end of promoters, and that orthologous core promoters across yeast species have conserved activities. Our results demonstrate the importance of core promoters in the quantitative study of gene regulation.

overall_design: Core promoter activity measured by FACS sorting cells and high throughput sequencing


title: Comprehensive analysis of microRNA expression in the human developing brain reveals microRNA-10 as a caudalizing factor

city: Lund

first: Malin

last: Parmar

address: BMC A11

department: Wallenberg Neuroscience Center

country: Sweden

institute: Lund University

summary: MicroRNAs (miRNAs) have been implicated in regulating multiple processes during brain development in various species. However, the function of miRNAs in human brain development remains largely unexplored. Here, we provide a comprehensive analysis of miRNA expression of regionalized neural progenitor cells derived from human embryonic stem cells and human fetal brain. We found that several miRNAs displaying both age-specific and region-specific expression patterns. Among these miRNAs we identified miR-10 to be specifically expressed in the human hindbrain and spinal cord, while absent from rostral regions. Gain-of-function experiments further demonstrated that miR-10 alone could drive caudalization of human neural progenitors cells. Together, these data confirms a role for miRNAs in establishing different human neural progenitor populations. This data set also provides a comprehensive resource for future studies investigating the functional role of different miRNAs in human brain development

overall_design: MicroRNA sequencing of 34 samples from human embryonic stem cells and human fetal brains.

first: Marie

middle: E

last: Jönsson

first: Jenny

last: Nelander Wahlestedt

last: Åkerblom

first: Agnete

last: Kirkeby

first: Johan

last: Jakobsson


title: De novo Reconstruction of Adipose Tissue Transcriptomes Reveals Novel Long Non-coding RNAs that Regulate Brown Adipocyte Development

city: Cambridge

first: Juan

last: Alvarez-Dominguez

state: MA

address: 7 Divinity Ave

department: Stem Cell and Regenerative Biology

institute: Harvard University

summary: Brown adipose tissue (BAT) protects against obesity by promoting energy expenditure via uncoupled respiration. To uncover BAT-specific long non-coding RNAs (lncRNAs), we used RNA-seq to reconstruct de novo transcriptomes of mouse brown, inguinal white, and epididymal white fat and identified ~1500 lncRNAs, including 127 BAT-restricted loci induced during differentiation and often targeted by key regulators PPARγ, C/EBPα and C/EBPβ. One of them, lnc-BATE1, is required for establishment and maintenance of BAT identity and thermogenic capacity. lnc-BATE1 inhibition impairs concurrent activation of brown fat and repression of white fat genes, and is partially rescued by exogenous lnc-BATE1 with mutated siRNA-targeting sites, demonstrating a function in trans. We show that lnc-BATE1 binds heterogeneous nuclear ribonucleoprotein U and that both are required for brown adipogenesis. Our work provides an annotated catalog for the study of fat depot-selective lncRNAs, available online, and establishes lnc-BATE1 as a novel regulator of BAT development and physiology.

overall_design: Total RNA profiles of BAT, iWAT and eWAT samples  were sequenced on the Illumina HiSeq2000 platform

first: Bingbing

last: Yuan


title: Expression and functions of long noncoding RNAs during human T helper cell differentiation

city: Nashville

first: Charles

last: Spurlock III

state: Tennessee

address: 1161 21st Avenue South

department: Medicine

institute: Vanderbilt University School of Medicine

summary: To improve our understanding of lncRNA expression in T cells, we used whole genome sequencing (RNA-seq) to identify lncRNAs expressed in human T cells and those selectively expressed in T cells differentiated under TH1, TH2, or TH17 polarizing conditions.  The majority of these lineage-specific lncRNAs are co-expressed with lineage-specific protein-coding genes.  These lncRNAs are predominantly intragenic with co-expressed protein-coding genes and are transcribed in sense and antisense orientations with approximately equal frequencies.  Further, genes encoding TH lineage specific mRNAs are not randomly distributed across the genome but are highly enriched in the genome in genomic regions also containing genes encoding TH lineage-specific lncRNAs.  Our analyses also identify a cluster of antisense lncRNAs transcribed from the RAD50 locus that are selectively expressed under TH2 polarizing conditions and co-expressed with IL4, IL5 and IL13 genes.  Depletion of these lncRNAs via selective siRNA treatment demonstrates the critical requirement of these lncRNAs for expression of the TH2 cytokines, IL-4, IL-5 and IL-13.  Collectively, our analyses identify new lncRNAs expressed in a TH lineage specific manner and identify a critical role for a cluster of lncRNAs for expression of genes encoding TH2 cytokines.

overall_design: Human peripheral blood mononuclear cells (PBMC) were cultured under TH1, TH2, and TH17 polarizing conditions. TH1, TH2, and TH17 primary and effector cultures were isolated and poly(A)+ and total RNA sequencing performed.

first: John

middle: T

last: Tossberg

first: Yan

last: Guo

first: Sarah

middle: P

last: Collier

first: Philip

last: Crooke III

first: Thomas

last: Aune


title: High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

city: Basel

first: Federico

last: Comoglio

state: Basel

address: Mattenstrasse, 26

department: Department of Biosystems Science and Engineering

country: Switzerland

institute: ETH Zurich

summary: At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, which genetic and chromatin features define metazoan replication start sites remains largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and show their ability to transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence specificity, mark and predict replication origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship between DNA topology, chromatin, transcription and replication initiation across metazoa.

overall_design: Our dataset consists of SNS-Seq profiles in Drosophila S2 and Bg3 cells. Small nascent leading strands (SNS) have been purified with an enhanced sensitivity SNS purification protocol. For standard SNS-Seq experiments (from pooled gradient fractions, denoted as "pool"), SNS were purified from two biological replicates. For single-fraction SNS-Seq experiments, two libraries were prepared for each fraction. The genomic coordinates of S2 and Bg3 replication origins are also provided.

first: Tommy

last: Schlumpf

first: Virginia

last: Schmid

first: Remo

last: Rohs

first: Christian

last: Beisel

first: Renato

last: Paro


title: Sequencing the cap-snatching repertoire of H1N1 influenza provides insight into the mechanism of viral transcription initiation

first: David

last: Koppstein

address: 9 Cambridge Center

institute: MIT

summary: The influenza polymerase cleaves host RNAs ~10–13 nucleotides downstream of their 5′ ends and uses this capped fragment to prime viral mRNA synthesis. To better understand this process of cap snatching, we used high-throughput sequencing to determine the 5′ ends of A/WSN/33 (H1N1) influenza mRNAs. The sequences provided clear evidence for nascent-chain realignment during transcription initiation and revealed a strong influence of the viral template on the frequency of realignment. After accounting for the extra nucleotides inserted through realignment, analysis of the capped fragments indicated that the different viral mRNAs were each prepended with a common set of sequences and that the polymerase often cleaved host RNAs after a purine and often primed transcription on a single base pair to either the terminal or penultimate residue of the viral template. We also developed a bioinformatic approach to identify the targeted host transcripts despite limited information content within snatched fragments and found that small nuclear RNAs and small nucleolar RNAs contributed the most abundant capped leaders. These results provide insight into the mechanism of viral transcription initiation and reveal the diversity of the cap-snatched repertoire, showing that noncoding transcripts as well as mRNAs are used to make influenza mRNAs.

overall_design: Sixteen datasets were generated, corresponding to template-switching 5’-RACE libraries of 1) each viral RNA, 2) a time-course of NS1 viral RNA, or 3) CIP-TAP libraries of NS1 and PB2. Libraries contain random barcodes of length 5 (NS1_TS, PB2_TS, NS1_5R, PB2_5R) or 9 (other datasets) at the start of each read, and at least three guanosine residues for the template-switching datasets. See methods of accompanying paper for further details. Samples were sequenced on an Illumina HiSeq 2000.

first: D

first: J

last: Ashour

last: Bartel


title: Identification and Expression Patterns of Novel Long Non-Coding RNAs in Neural Progenitors of the Developing Mammalian Cortex

city: Dresden

last: Calegari

address: Fetscherstr. 105

department: Center for Molecular and Cellular Bioengineering (CMCB)

institute: TU Dresden

summary: Long non-coding (lnc)RNAs play key roles in many biological processes. Elucidating the function of lncRNAs in cell type specification during organ development requires knowledge about their expression in individual progenitor types rather than in whole tissues. To achieve this during cortical development, we used a dual-reporter mouse line to isolate coexisting proliferating neural stem cells, differentiating neurogenic progenitors and newborn neurons and assessed the expression of lncRNAs by paired-end, high-throughput sequencing. We identified 379 genomic loci encoding novel lncRNAs and performed a comprehensive assessment of cell-specific expression patterns for all, annotated and novel, lncRNAs described to date. Our study provides a powerful new resource for studying these elusive transcripts during stem cell commitment and neurogenesis.

overall_design: mRNA profiles of Proliferating Progenitors, Differentiating Progenitors and Neurons from lateral cortex of E14.5 mouse embryos. Each cell type in three biological replicates.

first: Julieta

last: Aprea

first: Mathias

last: Lesche

first: Simone

last: Massalini

first: Silvia

last: Prenninger

first: Dimitra

last: Alexopoulou

first: Andreas

last: Dahl

first: Michael

last: Hiller


title: ELAVL1 modulates transcriptome-wide miRNA binding in murine macrophages

city: New Haven

first: Yi-Chien

last: Lu

state: Connecticut

address: 10 Amistad Street

department: Laboratory Medicine

institute: Yale Medical School

summary: Post-transcriptional gene regulation by miRNAs and RNA binding proteins (RBP) is important in development, physiology and disease. To examine the interplay between miRNAs and the RBP ELAVL1 (a.k.a. HuR), we mapped miRNA binding sites on a transcriptome-wide scale in WT and Elavl1 knockout murine bone marrow-derived macrophages. Proximity of ELAVL1 binding sites attenuated miRNA binding to transcripts and promoted gene expression. Transcripts that regulate angiogenesis and macrophage/ endothelial cross talk were preferentially targeted by miRNAs, suggesting that ELAVL1 promotes angiogenesis, at least in part, by antagonism of miRNA function.  We found that ELAVL1 antagonized binding of miR-27 to the 3’UTR of Zfp36 mRNA and alleviated miR-27-mediated suppression of the RBP ZFP36 (a.k.a. Tristetraprolin). Thus the miR-27-regulated mechanism synchronizes the expression of ELAVL1 and ZFP36.  This study provides a resource for systems-level interrogation of post-transcriptional gene regulation in macrophages, a key cell type in inflammation, angiogenesis and tissue homeostasis.

overall_design: Bone marrow derived macrpohges mRNA profiles of 7-day cultured wild type (WT) and Elavl1l-/- mouse bone marrow cells were generated by deep sequencing, with 4 biologic duplication, using Illumina GAII.

first: Sung-Hee

last: Chang

first: Markus

last: Hafner

first: Xi

last: Li

first: Thomas

last: Tuschl

first: Olivier

last: Elemento

first: Timothy

last: Hla


title: Dicer and Hsp104 function in a negative feedback loop to confer epigenetic robustness [RNA_correlations]

first: Tim

last: Roloff

address: Maulbeerstrasse 66

department: Functional Genomics

institute: FMI

summary: Epigenetic mechanisms can be influenced by environmental cues and thus evoke phenotypic variation. This plasticity can be advantageous for adaption, but also detrimental if not under tight control. Although having attracted considerable interest, it remains largely unknown if and how environmental cues such as temperature trigger epigenetic alterations. Using fission yeast, we demonstrate that environmentally induced discontinuous phenotypic variation is buffered by a negative feedback loop that involves the RNase Dicer and the protein disaggregase Hsp104. In the absence of Hsp104, Dicer accumulates in cytoplasmic inclusions and heterochromatin becomes unstable at elevated temperatures, an epigenetic state that is inherited for many generations after the heat stress. Dicer instead averts the toxic aggregation of a prionogenic protein. Our results highlight the importance of feedback regulation in building epigenetic memory and uncover Hsp104 and Dicer as homeostatic controllers that buffer environmentally induced stochastic epigenetic variation and toxic aggregation of prionogenic proteins.

overall_design: Various strains grown at 30°C or 37°C

first: Daniele

last: Oberti

first: Adriano

last: Biasini

first: Moritz

last: Kirschmann

first: Christel

last: Genoud

first: Rieka

last: Stunnenberg

first: Yukiko

last: Shimada

first: Marc

last: Bühler

first: Kirsten

last: Jacobeit


title: Native Elongating Transcript Sequencing Reveals Human Transcriptional Activity at Nucleotide Resolution

city: Boston

first: Stirling

last: Churchman

state: Massachusetts

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: Major features of transcription by human RNA polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates.

overall_design: 10 samples were analyzed. NET-seq data for HeLa S3 cells consisted in 2 WT biological replicates (HeLaS3_Rep1 and HeLaS3_Rep2), 1 treatment condition (HeLaS3_FP) and 1 control for the treatment condition (HeLaS3_DMSO). NET-seq data for HEK293T cells consisted in 2 WT biological replicates (HEK293T_Rep1 and HEK293T_Rep2). RNA-seq data for HeLa S3 cells consisted in the 3 cellular fractions in WT condition (HeLaS3_chromatin_RNAseq, HeLaS3_nucleoplasm_RNAseq and HeLaS3_cytoplasm_RNAseq). RNA-seq data for HEK 293T cells consisted in the cytoplasmic fraction in WT condition (HEK293T_cytoplasm_RNAseq)

first: L


title: Transcriptome Responses of the Host Spodoptera litura upon Spodoptera litura nucleopolyhedrovirus infection

status: Public on Apr 22 2015

city: Shanghai

first: Hanqi

last: Yin

state: Shanghai

address: libing road 151

institute: shanghai biochip corporation

summary: To reveal dynamic regulation of host gene expression and an overview of events in the infection cycle after SpltNPV infection

overall_design: To analyze cellular responses to SpltNPV infection, a S.litura cell line (SL221) was infected with wild-type baculovirus SpltNPV at a MOI of 1. RNAs were isolated at three time points through the infection cycle (6hpi, 12hpi and 18hpi) and also from control (mock infected) cells. The obtained cellular ranscriptome from the host S.litura was set for illumina strand specific RNA sequencing (RNA-seq) to analyze changes in host cell gene expression upon SpltNPV infection.

first: Qian

last: Yu

first: Jianliang

last: Liu

first: Qin

last: Wang

first: Dongling

last: Wen


title: Erosion of X chromosome inactivation in human pluripotent cells initiates with XACT coating and depends on a specific heterochromatin landscape

city: Paris

first: Jean-François

last: Ouimette

address: 35 rue Hélène Brion

department: UMR7216 Epigenetics and Cell Fate

institute: Université Paris Diderot

summary: Comparison of H3K9me3 and H3K27me3 distribution in homogeneous XaXi and XaXe H9 human embryonic stem cells and in IMR90 cells.

overall_design: Examination of 2 different histone modifications in 3 cell types.

first: Céline

last: Vallot

first: Claire

last: Rougeulle


title: Genomic Redistribution of GR Monomers and Dimers Mediates the Transcriptional Response to Exogenous Glucocorticoid In Vivo.

city: Neuherberg

first: Henriette

last: Uhlenhaut

state: Munich

address: Ingolstaedter Landstr 1

department: HDC

institute: Helmholtz

summary: Expression profiles of GRdim mutant macrophages (mouse, bone-marrow derived) treated with LPS for 6 hrs or with LPS (6hrs) + Dex (O/N 1uM). Identification of GR-regulated genes in response to LPS.

overall_design: 3 Grdim mutant macrophages samples treated with LPS (6hrs) and 3 Grdim mutant macrophage samples treated with LPS (6hrs) and Dex (overnight).

first: Nina

first: Norbert

last: Hübner


title: Neurotranscriptome profiles of four zebrafish strains

city: Omaha

first: Ryan

middle: Ying

last: Wong

state: NE

address: 6001 Dodge Street

department: Biology

institute: University of Nebraska at Omaha

summary: Zebrafish is a model system being used in a variety of basic research and biomedical studies. Understanding the neurotranscriptomic architecture will greatly facilitate and enhance interpretation of research projects. Studies have reported that there are strain and sex-specific behavioral variation particulary in response to stress and anxiety-inducing scenarios. Capitalizing on previously documented behavioral variation by strains and sex of zebrafish, this study seeks to understand the neurotranscriptomic mechanisms potentially underlying this variation. Through RNA-sequencing (4 biological replicates per strain further subdivided into 2 biological replicates per sex) we analyzed the whole-brain transcriptomic profiles of four strains of zebrafish and relate transcriptional differences to phenotypic differences (e.g. behavioral or morphological) of the strains. Using a balanced block design, all 16 samples were multiplexed and run across 16 lanes on an Illumina GAIIx. Resulting reads (approximately 52 million reads per biological replicate) were aligned to the Zv9 genome build. We subsequently performed differential gene expression analysis and weighted gene coexpression network analysis to identify genes and gene networks associated with a phenotype. The goal of the study is to identify neurotranscriptomic mechanisms underlying phenotypic (e.g. morphological, behavioral) variation in zebrafish.

overall_design: Through RNA-sequencing we quantified whole-brain transcriptome levels of protein-coding genes for four strains of zebrafish (AB, Scientific Hatcheries, High Stationary Behavior, and Low Stationary Behavior). Each line has 4 biological replicates (2 biological replicates for each sex). Each biological replicate is comprised of a pool of 10 same-sex and age-matched individuals. Using a balanced block design, the samples were mulitplexed and run across 16 lanes on an Illumina GAIIx. Reads that passed default quality control filters were aligned using GSNAP and quantified with HTSEQ. We used edgeR and WGCNA for subsequent differential gene expression and network analyses. qRT–PCR validation was performed using SYBR Green assays

middle: Y

first: John

last: Godwin


title: High-resolution nucleosome positioning from ATAC-seq chromatin accessibility data

city: Stanford

first: William

last: Greenleaf

state: CA

address: 279 Campus Drive West

institute: Stanford University

summary: We describe the NucleoATAC algorithm for high-resolution nucleosome positioning and occupancy determination using ATAC-seq.

sample_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae
Homo sapiens

overall_design: ATAC-seq was performed on Saccharomyces cerevisiae and Schizosaccaromyces pombe. Nucleosomes were called for both species as well as for the human GM12878 cell line using data from GSE47753.  Additionally, ATAC-seq was performed on S. cerevisiae undergoing an osmotic stress time-course and nucleosomes were called for each time point.

first: Alicia

last: Schep

first: Jason

last: Buenrostro

first: Sarah

last: Denny

first: Katja

last: Schwartz

first: Gavin

last: Sherlock


title: Telomerase subunit TERT regulates MYC stability independent of its role on telomeres

city: Singapore

first: Julius

last: Müller

address: 61 Biopolis Drive

department: IMCB

country: Singapore

institute: A*Star Singapore

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

overall_design: Refer to individual Series


title: Telomerase subunit TERT regulates MYC stability independent of its role on telomeres (ChIP-Seq)

summary: Constitutively active MYC and reactivated telomerase often co-exist in cancers. While the reactivation of telomerase is thought to be essential for replicative immortality, MYC, in conjunction with co-factors, confers several growth advantages to cancer cells. However, it is unclear which co-factors sustain elevated MYC activity in tumours . Here, we identify TERT, the catalytic subunit of telomerase, as a novel regulator of MYC stability in cancers. Binding of TERT to MYC stabilizes its levels on chromatin, contributing to either activation or repression of its target genes. Mechanistically, TERT regulates MYC ubiquitination and stability, and this effect of TERT is independent of its role on telomeres. Genetic inhibition and knocking out of TERT phenocopied the loss of MYC, resulting in reduced disease burden of early- and late-stage MYC-driven murine lymphomas. Conversly, the ectopic expression of TERT could substitute for reduced MYC in these functions. Finally we show that TERT null mice, unlike Terc null mice, show delayed onset of MYC induced lymphomagenesis. Accordingly, inhibiting TERT function in primary human leukemia cells blocked the expression of MYC targets, while Terc depletion had no effects .  Based on our data, we conclude that the re-expression of TERT, a direct MYC target in tumors, provides a feed-forward mechanism to potentiate MYC-dependent oncogenesis.

overall_design: ChIP was performed using anti-MYC (sc-764) in primary lymphoma cells from Eµ-Myc;Tert -/- mice, Eµ-Myc;Tert +/+ mice, or in P493 cells treated with shTert, P493 shTerc and shControl.

first: Cheryl

last: Koh

first: Ekta

last: Khattar

first: Shi

last: Leow

first: Chia

last: Liu

last: Muller

first: Wei

middle: X

last: Ang

first: Yinghui

last: Li

first: Guido

last: Franzoso

first: Shang

first: Ernesto

last: Guccione

first: Vinay

last: Tergaonkar


title: Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis (eRRBS)

city: Dundee

first: Kasper

middle: Dindler

last: Rasmussen

address: Dow Street

department: Division of Molecular, Cellular, and Developmental Biology (MCDB)

institute: School of Life Sciences, University of Dundee

type: Methylation profiling by high throughput sequencing

summary: DNA methylation is tightly regulated throughout mammalian development and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CpG islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO induced AML. Using this model, we show that the primary effect of Tet2 loss in pre-leukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner, but increase relative to population doublings. We confirm this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many downregulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation, and that it is the combined silencing of several tumor suppressor genes in TET2-mutated hematopoietic cells that contribute to increased stem cell proliferation and leukemogenesis.

overall_design: Enhanced Reduced Representation Bisulfite Sequencing (eRRBS) analysis of in vitro-grown hematopoietic cells transduced with AML1-ETO or MLL-AF9

middle: D

first: Guangshuai

last: Jia

first: Jens

middle: V

last: Johansen

first: Marianne

last: Pedersen

first: Nicolas

last: Rapin

first: Frederik

middle: O

last: Bagger

first: Bo

last: Porse

first: Olivier

last: Bernard

first: Jesper

last: Christensen

first: Kristian

last: Helin


title: mRNA profiling of wildtype, germline depleted, NMD mutant C. elegans whole worms and wildtype dissected gonads

city: Santa Cruz

first: James

middle: Matthew

last: Ragle

address: 1165 High Street Sinsheimer Labs 419

department: MCD Biology

institute: University of California-Santa Cruz

summary: Adjacent alternative 3’ splice sites, those separated by ≤18nt, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites.  Identification of the intron's 3' end depends upon sequence elements that define the branchpoint, polypyrimidine tract and terminal AG dinucleotide.  Starting with RNA-seq data from germline-enriched and somatic cell-enriched C. elegans samples, we identify hundreds of introns with adjacent alternative 3’ splice sites.  We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is mono-directional, with somatic cells preferring to splice at the distal 3' splice site and germline cells showing a distinct shift towards usage of the adjacent proximal 3' splice site.  Splicing patterns in somatic cells follow consensus rules of 3’ splice site definition, using sites with a short stretch of pyrimidines and an AG dinucleotide.  Splicing in germline cells occurs at proximal 3' splice sites that frequently lack a polypyrimidine tract or, occasionally, the AG dinucleotide.  We provide evidence that use of germline-specific proximal 3' splice sites is conserved across Caenorhabditis species.  We propose that divergent mechanisms exist between germline and somatic cells in determining an intron terminus at adjacent alternative 3’ splice sites.

overall_design: Examination of alternative splicing changes between germline- and somatic-cell enriched samples as well as nonsense-mediated decay mutants.

first: Sol

last: Katzman

first: Taylor

middle: F

last: Akers

first: Sergio

last: Barberan-Soler

first: Alan

last: Zahler


title: Genome-Wide Specificity of DNA-Binding, Gene Regulation, and Chromatin Remodeling by TALE- and CRISPR/Cas9-Based Transcription Factors

city: Durham

first: Timothy

last: Reddy

address: 2347 CIEMAS, 101 Science Drive

department: Department of Biostatistics & Bioinformatics

institute: Duke University

summary: Synthetic DNA-binding proteins have found broad application in gene therapies and as tools for interrogating biology. Engineered proteins based on the CRISPR/Cas9 and TALE systems have been used to alter genomic DNA sequences, control transcription of endogenous genes, and modify epigenetic states. Although the activity of these proteins at their intended genomic target sites have been assessed, the genome-wide effects of their action have not been extensively characterized.  Additionally, the role of chromatin structure in determining the binding of CRISPR/Cas9 and TALE proteins to their target sites and the regulation of nearby genes is poorly understood.  Characterization of the activity these proteins using modern high-throughput genomic methods would provide valuable insight into the specificity and off-target effects of CRISPR- and TALE-based genome engineering tools. We have analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators targeted to the promoters of two different endogenous human genes in HEK293T cells using a variety of high-throughput DNA sequencing methods. In particular, we assayed the DNA-binding specificity of these proteins and their effects on the epigenome. DNA-binding specificity was evaluated by ChIP-seq and RNA-seq was used to measure the specificity of these activators in perturbing the transcriptome. Additionally, DNase-seq was used to identify the chromatin state at target sites of the synthetic transcriptional activators and the genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these genome engineering technologies are highly specific in both binding to their promoter target sites and inducing expression of downstream genes when multiple activators bind to a single promoter. Moreover, we show that these synthetic activators are able to induce the expression of silent genes in heterochromatic regions of the genome by opening regions of closed chromatin and decreasing DNA methylation. Interestingly, the transcriptional activation domain was not necessary for DNA-binding or chromatin remodeling in these regions, but was critical to inducing gene expression. This study shows that these CRISPR- and TALE-based transcriptional activators are exceptionally specific. Although we detected limited binding of off-target sites in the genome and changes to genome structure, these off-target event did not lead to any detectable changes in gene regulation.  Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.

overall_design: HEK293T cells were transfected in triplicate with plasmids expressing synthetic transcription factors. The synthetic TFs were either (a) dCas9-VP64 fusion protein and a targeting guide RNA (gRNA), or (b) a TALE-VP64 fusion protein engineered to bind to a specific target site in the genome. As a control, cells were transfected with plasmids expressing GFP. After transfection, ChIP-seq was used to identify both on-target and off-target binding sites for the synthetic TFs.

first: Lauren

middle: R

last: Polstein

first: Pablo

last: Perez-Pinera

first: Dewran

last: Kocak

first: Christopher

middle: M

last: Vockley

first: Peggy

last: Bledsoe

first: Alexias

last: Safi

first: Lingyun

last: Song

first: Gregory

last: Crawford

first: Charles

middle: A

last: Gersbach


title: Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis

first: Kasper

last: Rasmussen


title: Small RNA mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

status: Public on Apr 21 2015

city: Ipswich

first: Guoqiang

last: Zhang

address: 240 County Road

institute: New England Biolabs Inc

summary: Mammalian cells contain copious amounts of RNA including both coding and non-coding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) binds directly to DNMT1 with high affinity. The binding of miRNAs, such as miR-155, leads to inhibition of DNMT1 enzyme activity. Exogenous miR-155 in cells induces aberrant DNA methylation of the genome, resulting in hypomethylation of low to moderately methylated regions. And small shift of hypermethylation of previously hypomethylated region was also observed. Furthermore, hypomethylation led to activation of genes. Based on these observations, we propose that overexpression of specific miRNAs in human cancer may lead to aberrant DNA methylation and altered gene-expression.

overall_design: Examine of the DNA methylation and mRNA profile of HCT 116 cells transfected by random 23-mer or miR-155 RNA

first: Sriharsa

last: Pradhan


title: TAF10 interacts with the GATA1 transcription factor and controls mouse erythropoiesis

city: Leuven

first: Petros

last: Papadopoulos

address: Herestraat 49

department: Center for Human Genetics

country: Belgium

institute: KU Leuven

summary: We have ablated TAF10 in the erythroid compartment only by crossing the TAF10lox mice with the EpoR-Cre mice and we have studied the development of the erythroid cells in vivo. TAF10 ablation led to embryonic death at E13.5 while at E12.5 there was a clear developmental defect which was reflected in the transcriptional profile of the fetal liver cells. Gata1-target genes were mostly affected and were responsible for the lethal phenotype.

overall_design: mRNA from E12.5 fetal livers of TAF10lox/KO:EpoR-Cre+/- (TAF10KO) mice, TAF10HET and WT mice was profiled by NGS (Illumina).

first: Laszlo

last: Tora


title: Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis (MeDIP-seq)

overall_design: 5hmC-DIP-seq analysis for distribution of 5hmC in in vitro-grown hematopoietic cells transduced with AML1-ETO

first: Guangshuai

last: Jia

first: Jens

last: Johansen

first: Marianne

last: Pedersen

first: Nicolas

last: Rapin

first: Frederik

last: Bagger

first: Bo

last: Porse

first: Olivier

last: Bernard

first: Jesper

last: Christensen

first: Kristian

last: Helin


title: Sex bias and maternal contribution to gene expression divergence in Drosophila blastoderm embryos

city: LYON

first: Mathilde

last: Paris

address: Faculte des sciences de Luminy, case 907

department: IGFL

institute: CNRS

summary: Early embryogenesis is a unique developmental stage where genetic control of development is handed off from mother to zygote. Yet the contribution of this transition to the evolution of gene expression is poorly understood. Here we study two aspects of gene expression specific to early embryogenesis in Drosophila: sex-biased gene expression prior to the onset of canonical X chromosomal dosage compensation, and the contribution of maternally supplied mRNAs. We sequenced mRNAs from individual unfertilized eggs, precisely staged and sexed blastoderm embryos and compared levels between D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. First, we find that mRNA content is highly conserved for a given stage and that studies relying on pooled embryos may systematically overstate the degree of gene expression divergence. Unlike studies done on larvae and adults, where most species show a larger proportion of genes with male-biased expression, we find that transcripts in Drosophila embryos are largely female-biased in all species, likely due to incomplete dosage compensation prior to the activation of the canonical dosage compensation mechanism. The divergence of sex-biased gene expression across species is observed to be often due to a decrease of expression, the most drastic example of which is the overall reduction of male expression from the neo-X chromosome in D. pseudoobscura, leading to a pervasive female-bias on this chromosome. We see no evidence for a faster evolution of expression on the X chromosome in embryos (no “faster X” effect), unlike in adults and contrary to a previous study on pooled non-sexed embryos. Finally, we find that most genes are conserved in regard to their maternal or zygotic origin of transcription, and present evidence that differences in maternal contribution to the blastoderm transcript pool may be due to species-specific divergence of transcript degradation rates

sample_organism:
Drosophila melanogaster
Drosophila pseudoobscura
Drosophila virilis
Drosophila yakuba

overall_design: We sequenced mRNA from D. melanogaser, D. yakuba, D. pseudoobscura and D. virilis single unfertilized eggs (1 to 2 per species) and from both single female and male embryos (3 per sex per species).

first: Jacqueline

last: Villalta

first: Michael

middle: B

last: Eisen

first: Susan

last: Lott


title: Dicer and Hsp104 function in a negative feedback loop to confer epigenetic robustness

first: Tim

last: Roloff


title: SF3B1 association with chromatin determines splicing outcomes

city: Tel- aviv

first: Gil

last: Ast

state: Israel

address: Ramat Aviv

department: Human Molecular Genetics & Biochemistry

institute: Tel-Aviv University

summary: Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. Part of the explanation probably lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicated that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses revealed that SF3B1 is specifically bound to nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affected the splicing of these exons similarly to inhibition of SF3B1 expression. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure.

overall_design: MNase-seq on Input and SF3B1 pull-down, mRNA-seq on control and SF3B1 si-RNA treated cells as well as on TSA (Trichostatin A) treated and untreated cells.

first: Nir

last: Kfir

first: Galit

last: Lev-Maor

first: Ohad

last: Glaich

first: Adi

last: Alajem

first: Arnab

last: Datta

first: Sze-Sui

last: Kwan

first: Eran

last: Meshorer


title: Dicer and Hsp104 function in a negative feedback loop to confer epigenetic robustness [RNA_profiles]

city: Basel

overall_design: wt_spb75 grown at 30°C or 37°C

first: Daniele

last: Oberti

first: Adriano

last: Biasini

first: Moritz

last: Kirschmann

first: Kirsten

last: Jacobeit

first: Christel

last: Genoud

first: Rieka

last: Stunnenberg

first: Yukiko

last: Shimada

first: Marc

last: Bühler


title: High-throughput sequencing of pluripotent cells

city: Salt Lake City

first: Edward

middle: J

last: Grow

state: UT

address: 2000 Circle of Hope Drive

department: Oncology

institute: University of Utah

summary: We report HERV-K rec iCLIP-seq binding data, ribosome profiling data, and RNA-seq from ELF1 naïve hESC and RNA-seq from NCCIT cells.

overall_design: HERV-K Rec iCLIP-seq: 2 replicates in NCCIT. Ribosome profiling: 4 replicates each of Rec-overexpressing NCCIT vs. control NCCIT; RNAseq: 3 replicates each of HERV-K Rec siRNA vs. control siRNA in NCCIT; RNA-seq: 3 replicates each of ELF1 naïve hESC vs. primed hESC.

first: Joanna

last: Wysocka


title: Genome-wide transcriptome analysis during dehydration in leaf tissues of Boea hygrometrica

city: Beijing

first: Lihong

last: Xiao

address: No.105 North Road of West 3rd-ring Road.Haidian District

institute: Capital Normal University

summary: Gene transcript abundances were analyzed with samples taken from hydrated, moderate dehydration (70% RWC) and desiccated (10% RWC) leaf tissues of resurrection plant species B. hygrometrica by using RNA-Seq. Totally, 9888 genes were identified as differentially expressed genes.The results provided insight for exploring the mechanisms of desiccation tolerance.

overall_design: Examination of mRNA transcript abundances in hydrated, dehydrating to 70% RWC and dehydrating to 10% RWC leaf tissues, for each treatment, three biological replicates were included.


title: mRNA profiling reveals determinants of trastuzumab efficiency in HER2-positive breast cancer

city: Göttingen

first: Silvia

last: von der Heyde

address: Humboldtalle 32

department: Medical Statistics

institute: University Medical Center Göttingen

summary:
The intention was to detect genes that are determining trastuzumab efficiency in HER2-positive breast cancer cell lines with different resistance phenotypes.
While BT474 should be sensitive to the drug treatment, HCC1954 is expected to be resistant due to a PI3K mutation.
The cell line BTR50 has been derived from BT474 and was cultured to be resistant as well.
Based on RNA-Seq data, we performed differential expression analyses on these breast cancer cell lines with and without trastuzumab treatment.
In detail, five separate tests were performed, namely resistant cells vs. wild type, i.e. HCC1954 and BTR50 vs. BT474, respectively, and untreated vs. drug treated cells.
The significant genes of the first two tests should contribute to resistance.
The significant genes of the test BT474 vs. its drug treated version should contribute to the trastuzumab effect.
To exclude false positives from the combined gene set (#64), we removed ten genes that were also significant in the test BTR50 vs. its drug treated version.
This way we ended up with 54 genes that are very likely to determine trastuzumab efficiency in HER2-positive breast cancer cell lines.

overall_design: mRNA profiles of human breast cancer cell lines were generated by deep sequencing using Illumina HiSeq 2000. The cell lines BT474 and HCC1954 were analyzed with and without trastuzumab treatment. HCC1954 is known to be trastuzumab resistant. Additionally, the cell line BTR50 was generated as resistant version of BT474, and was analyzed with and without trastuzumab as well.

first: Steve

last: Wagner

first: Alexander

last: Czerny

first: Manuel

last: Nietert

first: Fabian

last: Ludewig

first: Gabriela

last: Salinas-Riester

first: Tim

last: Beißbarth


title: Optimal codons in Tremella fuciformis end in C/G, a strong difference with known Tremella species

status: Public on Apr 20 2015

city: Fuzhou

first: Baogui

last: Xie

state: Fujian

address: Shangdian Road

department: Mycological Research Center

institute: mrcfafu

summary: Tremella fuciformis is a popular edible fungus with fruiting bodies that can be produced in large quantities at low costs, while it is easy to transform and cultivate as yeast. This makes it an attractive potential bioreactor. Enhanced heterologous gene expression through codon optimization would be useful, but until now codon usage preferences in T. fuciformis remain unknown. The only available genome of a species in the genus Tremella was that of Tremella mesenterica. To precisely determine the preferred codon usage of T. fuciformis we sequenced the genome of strain Tr26 resulting in a 24.5 Mb draft genome with 10,040 predicted genes. 3288 of the derived predicted proteins matched the UniProtKB/Swiss-Prot databases with 40% or more similarity. Corresponding gene models of this subset were subsequently optimized trough repetitive comparison of alternative start codons and selection of best length matching gene models. For experimental confirmation of gene models, 96 random clones from an existing T. fuciformis cDNA library were sequenced, generating 80 complete CDSs. Calculated optimal codons (RSCU and RFCU values) for the 3288 predicted and the 80 cloned CDSs were highly similar, indicating sufficient accuracy of predicted gene models for codon usage analysis. T. fuciformis showed a strong preference for C and then G (C+G; 66.4 %) at the third base pair position of used codons, while average GC content of predicted genes was slightly higher (58.5 %) than he total genome sequence average (55.9%). Most frequently used codons (optimal codons) all ended in C or G except for one (Ter; AGU), and an increased frequency of C ending codons was observed in genes with higher expression levels. Surprisingly, the preferred codon usage in T. fuciformis strongly differed from T. mesenterica (same genus) and C. neoformans (same family). Instead, optimal codon usage was similar to more distant related species such as Ustilago maydis and Neurospora crassa. Despite much higher overall sequence homology between T. fuciformis and T. mesenterica only 7 out of 21 optimal codons were equal, whereas T. fuciformis shared up to 20 out 21 optimal codons with other species. Clearly, codon usage in Tremella can differ largely and should be estimated for individual species. The precise identification of optimal and high expression related codons is therefore an important step in the development of T. fuciformis as a bioreactor system.

overall_design: Using the Total RNA was extracted from yeast-like cells of T. fuciformis Tr21 in their exponential growth stage, and double-stranded cDNA was synthesized for tag library construction and digital gene expression tag (DGE) sequencing at BGI Tech Solutions Co., Ltd (Shenzhen, China). Image analysis, base calling, extraction of tags, and tag counting were conducted using the Illumina pipeline. Clean tags were mapped to predicted gene models of the draft genome with a mismatch tolerance of 1bp. The number of tags for each CDS was calculated and then normalized to TPM (number of transcripts per million clean tags)  digital gene expression (DGE)

first: Youjin

last: Deng

first: Xiaoxing

last: Huang

first: Banzhan

last: Ruan

first: Arend

last: Frans van Peer

first: Yuji

last: Jiang


title: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors

city: Dallas

first: James

middle: Francis

last: Amatruda

state: TX

address: 5323 Harry Hines Blvd

department: Pediatrics, Internal Medicine, and Molecular BIology

institute: UT Southwestern

summary: Through whole-exome sequencing we identified somatic missense mutations in DICER1 and DROSHA in Wilms tumor, a childhood kidney cancer.  DICER1 and DROSHA are key enzymes in the microRNA biogenesis pathway.  To determine the effect of these mutations on microRNA expression, we prepared small RNAs from Wilms tumors and used next-generation sequencing to determine the expression levels of microRNAs in the tumors.

overall_design: Comparison of miRNA expression in tumors with and without mutations in DICER1 or DROSHA.

first: Joshua

last: Mendell


title: A dominant role for the methyl-CpG-binding protein Mbd2 in controlling Th2 induction by dendritic cells

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

institute: Centre for Immunity, Infection and Evolution


title: Allogeneic mature human dendritic cells generate superior alloreactive regulatory T cells in the presence of  IL-15

city: Rotterdam

first: Nicolle

last: Litjens

address: Wytemaweg 80

department: Internal Medicine

country: Netherlands

institute: Erasmus MC

summary:
Introduction: Expansion of antigen (Ag)-specific natural occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloAg-specific nTregs.
Methods: A highly enriched nTreg-fraction (CD4+CD25brightCD127- T cells) was alloAg-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDC) or peripheral blood mononuclear cells (PBMC). The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the TSDR of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4 and cytokines. In addition, the antigen-specific suppressive capacity of these expanded nTregs was tested.
Results: Allogeneic mature moDC and skin-derived DC were superior in inducing nTreg-expansion compared to immature moDC or PBMC in an HLA-DR and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2, could facilitate optimal mature moDC-induced nTreg-expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloAg-induced proliferation without significant suppression of completely HLA-mismatched-Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the TSDR within the FOXP3 gene and highly expressed of FOXP3, HELIOS and CTLA4.  A minority of the expanded nTregs produced IL-10, IL-2, IFN-g and TNF-a but very few IL-17 producing nTregs were found. Next generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs.
Conclusions: Human allogeneic mature moDC are highly efficient stimulator cells, in presence of exogenous IL-15, for expansion of stable alloAg-specific nTregs with superior suppressive function.

overall_design: Four different batches of highly pure regulatory T cells (all from the same donor) were expanded in two different ways, and compared to non-expanded samples.

middle: H

first: Karin

last: Boer

first: Joke

last: Zuijderwijk

first: Mariska

last: Klepper

first: Annemiek

last: Peeters

first: Errol

last: Prens

first: Wenda

last: Verschoor

first: Rens

last: Kraaijeveld

first: Zeliha

last: Ozgur

first: Mirjam

middle: C

last: van den Hout-van Vroonhoven

first: Wilfred

last: van IJcken

first: Carla

last: Baan

first: Michiel

middle: G

last: Betjes


title: A dominant role for the methyl-CpG-binding protein Mbd2 in controlling Th2 induction by dendritic cells [ChIP-Seq]

summary: Dendritic cells (DCs) direct CD4+ T cell differentiation into distinct T helper (Th) subsets that are vital for protection against diverse types of infection.  However, the mechanisms employed by DCs to initiate Th2 responses, which are important for immunity to helminth infection as well as being a major contributor to allergic disease, remain poorly understood.  We demonstrate a key role for methyl-CpG-binding domain-2 (Mbd2), a protein that links DNA methylation to repressive chromatin structure, in regulating expression of a range of genes that are associated with optimal DC activation and function. In the absence of Mbd2, DCs displayed reduced phenotypic activation and a dramatically impaired ability to promote Th2 immunity against either helminths or allergens, while their induction of Th1/17 responses remained intact. These data reveal a novel epigenetic mechanism that is integral to DC promotion of CD4+ T cell responses, particularly in Th2 settings, and identify methyl-CpG-binding proteins and the genes that they control as potential therapeutic targets for type-2 inflammation.

overall_design: H3K9,K14ac (H3ac) occupancy was analysed by ChIP-Seq for control (wt) or Mbd2 -/- (DEL) dendritic cells (DC). Input DNA for each cell line was also sequenced alongside.

first: Peter

last: Cook

first: Heather

last: Owen

first: Aimée

last: Deaton

first: Jessica

last: Borger

first: Sheila

middle: L

last: Brown

first: Thomas

last: Clouaire

first: Gareth-Rhys

last: Jones

first: Lucy

first: Rachel

last: Lundie

first: Angela

middle: K

last: Marley

first: Vicky

last: Morrison

first: Alexander

middle: T

last: Phythian-Adams

first: Elisabeth

last: Wachter

first: Lauren

last: Webb

first: Tara

middle: E

last: Sutherland

first: Graham

last: Thomas

first: John

last: Grainger

first: Jim

last: Selfridge

first: Andrew

middle: N

last: McKenzie

first: Judith

last: Allen

first: Susanna

last: Fagerholm

first: Rick

last: Maizels

first: Adrian

last: Bird

middle: S

last: MacDonald


title: RNA-sequencing of unstressed and pressure-overloaded C57 mouse hearts with single or combined ablation of PKCdelta and PKCepsilon

city: Indianapolis

first: Scot

last: Matkovich

state: IN

address: Lilly Corporate Center

institute: Eli Lilly and Company

summary: Combinatorial ablation of the major cardiac-expressed novel protein kinase C isoforms, PKCdelta and PKCepsilon, in murine cadiac myocytes unmasked functionally redundant growth-limiting effects of these kinases in hemodynamically stressed adult hearts and during normal embryonic cardiac development, mediated in part by shared transcriptional de-repression of ERK and periostin signaling.

overall_design: 14 cardiac polyA+-RNA profiles from unstressed, 12 week-old C57BL/6J-background mice (4 wild-type, 3 postnatal cardiac PKCdelta ablation, 3 germline PKCepsilon knockout, 4 combined knockouts) and 15 profiles generated 4 weeks after TAC induction (4 wild-type, 3 PKCdelta, 4 PKCepsilon, 4 combined knockout) were generated on Illumina HiSeq 2000 instruments.

first: Gerald

middle: W

last: Dorn


title: A subcutaneous adipose tissue-liver axis in the control of hepatic gluconeogenesis

status: Public on Apr 17 2015

city: La Jolla

first: Ruth

last: Yu

address: 10010 N Torrey Pines Rd

department: Gene Expression Lab

institute: Salk Institute

summary: We performed RNA sequencing analysis of hepatic gene expression a few hours after amlexanox treatment, and identified over 1700 differentially expressed genes. Pathway analysis of these differentially regulated genes revealed that the top two most enriched pathways were the adipocytokine signaling pathway and the Jak-STAT signaling pathway.

overall_design: RNA-seq analysis of hepatic gene expression was used to identify differentially expressed genes in response to Amlexanox treatment.

first: Shannon

last: Reilly

first: Alan

last: Saltiel

first: Ronald

last: Evans


title: Opposing roles for the lncRNA Haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation

city: Rochester

first: jinlong

last: lu

state: NY

address: 213 Hutchinson Hall

institute: University of Rochester

summary: Long noncoding RNAs (lncRNAs) have been implicated in controlling various aspects of embryonic stem cell (ESC) biology, although the functions of specific lncRNAs, and the molecular mechanisms through which they act, remain unclear. Here, we demonstrate discrete and opposing roles for the lncRNA transcript Haunt and its genomic locus in regulating the HOXA gene cluster during ESC differentiation. Reducing or enhancing Haunt expression, with minimal disruption of the Haunt locus, led to up- or down-regulation of HOXA genes, respectively. In contrast, increasingly large genomic deletions within the Haunt locus attenuated HOXA activation. The Haunt DNA locus contains potential enhancers of HOXA activation, whereas Haunt RNA acts to prevent aberrant HOXA expression. This work reveals a multi-faceted model of lncRNA-mediated transcriptional regulation of the HOXA cluster, with distinct roles for a lncRNA transcript and its genomic locus, while illustrating the power of rapid CRISPR/Cas9-based genome editing for assigning lncRNA functions.

overall_design: All RNA-seq(s) were designed to reveal the differentially expressed genes among different stages of ESCs differentiation, or differentially expressed genes between wild-type or Haunt or HOXA mutant cells during RA-induced differentiation. All ChIRP-Seq were used to reveal the DNA or RNA targets of Haunt before or after RA treatment.

first: Yafei

last: Yin

first: Jinlong

last: Lu

first: Xiaohua

last: Shen


title: Integrative Genomic Analysis Reveals Widespread Enhancer Regulation by p53 in Response to DNA Damage

status: Public on Apr 18 2015

city: Kansas City

first: Scott

last: Younger

state: MO

address: 2401 Gillham Road

institute: Children's Mercy Kansas City

summary: The tumor suppressor p53 has been studied extensively as a direct transcriptional activator of protein-coding genes.  Recent studies, however, have shed light on novel regulatory functions of p53 within noncoding regions of the genome.  Here, we use a systematic approach that integrates transcriptome-wide differential expression analysis, genome-wide p53 binding profiles, chromatin state maps, and additional genomic features to characterize the global regulatory roles of p53 in response to DNA damage in both human and mouse fibroblast models.  In addition to known p53 targets, we identify many previously unappreciated mRNAs and long noncoding RNAs that are regulated by p53.  Moreover, we find that p53 binding events occur predominantly within enhancer elements in both human and mouse systems.  The ability to modulate enhancer activity offers an additional layer of complexity to the p53 network and greatly expands the diversity of genomic elements that are directly regulated by p53.

overall_design: Human and Mouse fibroblasts cultured in the presence or absence of doxorubicin followed by RNA-Seq (Human:2 cell lines, each condition in duplicate; Mouse:MEF cell line,each condition in triplicate) and p53 ChIP-Seq (Human:2 cell lines, input and IP for each; Mouse:MEF cell line, input and IP)

first: John

last: Rinn


title: Phosphorylation events in the Multiple Gene Regulator of Group A Streptococcus (Mga) Significantly Influences Global Gene Expression and Virulence.

city: Houston

first: Randall

last: Olsen

address: 6565 Fannin Street; MS:B250

department: Pathology and Genomic Medicine

institute: Houston Methodist Research Institute

summary: We sought understand the molecular mehcanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS through whole transcriptome analysis of strains with phosphorylation mimicking substitutions in key histidine residues of Mga.

overall_design: There were 6 strains analyzed, each in triplicate replicates: 1)wild-type GAS, 2)mga deletion strain 3)mga with alanine at H207 4)mga with aspartate at H207, 5) mga with alanine at H273 6)mga with aspartate at H273

first: Misu

last: Sanson

first: Muthiah

last: Kumaraswami


title: Histone modifications induced by MDV infection at early cytolytic and latency phases

city: Bethesda

first: Apratim

last: Mitra

state: MD

address: 6 Center Dr Building 6B Room 2B206

department: Division of Developmental Biology

institute: NICHD

summary:
Background: Marek’s disease (MD) is a highly contagious, lymphomatous disease of chickens induced by a herpesvirus, Marek’s disease virus (MDV) that is the cause of major annual losses to the poultry industry. MD pathogenesis involves multiple stages including an early cytolytic phase and latency, and transitions between these stages are governed by several host and environmental factors. The success of vaccination strategies has led to the increased virulence of MDV and selective breeding of naturally resistant chickens is seen as a viable alternative. While multiple gene expression studies have been performed in resistant and susceptible populations, little is known about the epigenetic effects of infection.
Results: In this study, we investigated temporal chromatin signatures induced by MDV by analyzing early cytolytic and latent phases of infection in the bursa of Fabricius of MD-resistant and –susceptible birds. Major global variations in chromatin marks were observed at different stages of MD in the two lines. Differential H3K27me3 marks were associated with immune-related pathways, such as MAP kinase signaling, focal adhesion and neuroactive ligand receptor interaction, and suggested varying degrees of silencing in response to infection. Immune-related microRNAs, e.g. gga-miR-155 and gga-miR-10b, bore chromatin signatures, which suggested their contribution to MD-susceptibility. Finally, several members of the focal adhesion pathway, e.g. THBS4 and ITGA1, showed marked concordance between gene expression and chromatin marks indicating putative epigenetic regulation in response to MDV infection.
Conclusions: Our comprehensive analysis of chromatin signatures, therefore, revealed further clues about the epigenetic effects of MDV infection although further studies are necessary to elucidate the functional implications of the observed variations in histone modifications.

overall_design: Total of 32 samples analyzed; 2 histone modifications - H3K4me3 and H3K27me3 x 2 chicken lines with varying resistance to MD - L63 and L72 x 2 time-points of disease progression - 5 and 10 days post infection x 2 conditions - infected and control x 2 biological replicates

first: Yanghua

last: He

first: Jiuzhou

last: Song


title: Functional and Mechanistic Studies of XPC DNA-Repair Complex as Transcriptional Coactivator in Embryonic Stem Cells

city: Berkeley

first: Robert

last: Tjian

address: University of California, Berkeley 450 Li Ka Shing #3370

department: MCB

institute: UC Berkeley

summary: We recently identified the DNA repair complex XPC-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4 and SOX2 transcriptional activation. Here we investigate genome-wide the role of SCC in murine ESCs by mapping regions bound by its RAD23B subunit in wild type and Xpc-/- ESCs, and analyzing transcriptional profiles of SCC-depleted ESCs.

overall_design: RAD23B ChIP-seq in wild type murine ESCs was performed in two replicates; normal IgG were used as controls. RAD23B ChIP-seq in Xpc-/- mESCs was compared to normal IgG. Antibodies to immunoprecipitate RAD23B were raised in guinea pigs against the 108-177 peptide of murine RAD23B protein (NP_033037). Anti-sera were affinity-purified. For RNA-seq, we compared wild type JM8.N4 cells with either Rad23b-/- cells, Rad23b-/-Xpc KD1 or Rad23b-/-Xpc KD2 cells. Xpc was knocked down with two independent shRNAs (KD1 and KD2).

first: Claudia

last: Cattoglio

first: Ivan

last: Grubisic


title: Topologically-associating domains and their long-range contacts are established during early G1 coincident with the establishment of the replication timing program

city: Tallahassee

first: David

middle: M.

last: Gilbert

state: Florida

address: 319 Stadium Drive

institute: Florida State University

summary: This study uncovers a temporal hierarchy of chromatin re-organization during G1 that is linked to the developmental and temporal control of replication timing, revealing a novel link between development and genome organization.

overall_design: Analysis of time-course 4C-seq for several baits to study dynamic changes in chromatin organization during early G1

first: Vishnu

last: Dileep

first: Ferhat

last: Ay

first: Jiao

last: Sima

first: William

last: Noble


title: RAR/RXR binding dynamics distinguishes pluripotency from differentiation-associated cis-regulatory elements

city: Villeurbanne

first: Philippe

last: Veber

address: 16 rue Raphael Dubois

institute: CNRS

summary: In mouse embryonic cells, a retinoic acid (RA) stimulation triggers a massive change of gene expression leading the pluripotent, proliferating cells to a lineage-specific differentiation process. The retinoic acid receptor (RAR) plays a key role in this response by inhibiting pluripotency-maintaining genes and simultaneously activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 hours of the RA-induced Primitive Endoderm differentiation process in F9 embryonic carcinoma cells. We detected significantly more RAR/RXR binding regions than previous studies and identified among them a handful of typical binding intensity patterns during differentiation. We demonstrate that these patterns are correlated with the coincidental binding of essential transcription factors (TFs) for pluripotency maintenance or PrE differentiation of embryonic stem (ES) cells, as well as the presence of variants of RAR binding motifs. Most importantly, early-bound regions coincide with pluripotency-associated transcription factor binding in ES (like Pou5f1, Sox2, Esrrb and Nr5a2) and display an increased frequency of the DR0 type RAR binding motifs; late-bound sites are associated to the PrE marker Sox17 and are enriched in the canonical DR5 binding motif. Our data offer an unprecedently detailed view on the action of RA in triggering pluripotent cell differentiation. Altogether, this work sheds light on the relocation of RAR/RXR binding sites throughout differentiation, and shows how RAR/RXR progressively shift from DR0 enriched regions, which were specifically identified in undifferentiated models, to canonical RAR binding sites containing loci.

overall_design: Time course (0, 2, 6, 12, 24, 48h) after stimulation of F9 cultured cells by retinoic acid: PanRAR and PanRXR ChIP-seq at 0, 2, 24 and 48h (no replicate); WCE-seq at 0h (no replicate); mRNA-seq at 0, 6, 12, 24, 48h (2 replicates except for time 0h, 4 replicates). Additionally, a control time course (culture in DMSO) sampled at 24 and 48h (no replicates).

first: Amandine

last: Chatagnon

first: Justin

last: Bedo

first: Valérie

last: Morin

first: Gérard

last: Triqueneaux

first: Marie

last: Sémon

first: Vincent

last: Laudet

first: Florence

last: d'Alché-Buc

last: Benoit


title: Mcm2-7 is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of its DNA Gate

city: Durham

last: MacAlpine

state: NC

address: Box 3813 DUMC

department: Pharmacology and Cancer Biology

institute: Duke University Medical Center

summary: Genome wide characterization of Mcm2-7 localization and origin activity for mrc1 and mcm mutant alleles.

overall_design: Samples used in this study: alpha-factor arrested samples  Mcm2p ChIP:  wild type (UPY493) 1x  mcm2DENQ (UPY524) 1x    Mcm2-7 ChIP:  wild type (UPY493) 1x  mcm2DENQ (UPY524) 1x    Hu Arrested samples  BrdU Chip:  wild type (UPY493) 2x  mcm2DENQ (UPY524) 2x    Mcm2-7 ChIP:  wild type (UPY493) 2x  mcm2DENQ (UPY524) 2x  mrc1∆ (UPY722) 2x  Rationale. To assay the effects of origin activation, cultures were arrested in G1, released into BrdU containing media, and subsequently processed for ChIP.  To assay the efficacy of late firing origins, cultures were synchronized in G1 as above, released into Hu + BrdU containing media, and processed for ChIP      Experimental Outline. Chromatin immunoprecipitation (ChIP) was performed as described (Aparicio et al., 1997; Katou et al., 2003) using yeast engineered for BrdU uptake (Vernis et al., 2003).  Sequencing libraries were generated from the above immunoprecipitated DNA using the Illumina sample prep kit, multiplexed, and sequenced on a GAII Illumina sequencer.      Genome Assembly. For each sample sequenced reads were aligned to the S. cerevisiae S288C reference genome (SacCer2, SGD June 2008) (Kent et al., 2002) using MAQ (Li et al., 2008) allowing up to three mismatched bases. Once mapped, only those reads with a Phred quality score of 35 or greater were used for downstream analysis. For each experiment, reads were  binned across the genome, and RPKM (reads per kilobase per million mappable reads) was calculated for each bin.  For all of the BrdU ChIP-Seq experiments, bins of 5000 bp stepping every 1250 bp were used, and for all the Mcm ChIP-Seq experiments, bins of 1000 bp stepping every 250 bp were used. All in silico analyses were done in the R programming environment using the Bioconductor suite of packages (Gentleman et al., 2004; RDevelopmentCoreTeam, 2010). Experimental replicates were quantile normalized and combined by calculating the mean score within each bin.    Peak determination. For each bin along a chromosome, a probability was assigned according to that chromosome’s empirical cumulative distribution of RPKM. A threshold was determined, and bins with a probability greater than or equal to the cutoff were taken as peaks (the ranges of overlapping peaks were merged such that the resulting peaks represented the union of all enriched bins).  For BrdU ChIP-Seq in HU arrested cells, the thresholds used were as follows: WT strains p ≥ 0.95, Mrc1 p ≥ 0.75,  and Mcm2DENQ p ≥0.75. For ChIP-Seq experiments in G1 arrested cells, a threshold of p ≥ 0.96 was used. Replicates were combined by taking the intersection of peak calls.  When possible, peaks were assayed for concordance against analogous data previously reported (Crabbe et al., 2010).

first: Feng-Ling

last: Tsai

first: Joseph

last: Prinz

first: Heather

first: Anthony

last: Schwacha