title: The Immune Strategy and Stress Response of the Whitefly, Bemisia tabaci, to an Orally Delivered Bacterial Pathogen

city: Hangzhou

first: Xiaowei

last: Wang

address: 866 Yuhangtang Road

department: Insect Sciences

institute: Zhejiang University

summary: The whitefly, Bemisia tabaci, a notorious agricultural pest, has complex relationships with diverse microbes. It recognizes and degrades pathogens, as other insects do, and also relies on endosymbionts for its survival. Both types of interaction have received great attention, because of their potential importance in developing novel whitefly control technologies. The recent developments in RNA-seq technology allows us to perform a comprehensive investigation of a whitefly’s defense responses after it has ingested the pathogen, Pseudomonas aeruginosa. Compared to uninfected whiteflies, 6 and 24 hour post-infected (hpi) whiteflies showed 1,348 and 1,888 differentially expressed genes, respectively. Functional analysis highlighted the involvement of mitogen associated protein kinase (MAPK) pathway in host-defense regulation. Three knottin-like antimicrobial peptide genes and several components of the humoral and cellular immune response were also activated, indicating that key immune elements recognized in other insect species are also important for the host response of B. tabaci. Our data also suggest that intestinal stem cell mediated epithelium renewal might be an important component of the whitefly’s defense against oral bacterial infection. In addition, we also show stress responses to be an essential component of the defense system. We identify for the first time the key immune-response elements utilized by B. tabaci against bacterial infection. This provides a framework for future research into the complex interactions between whiteflies and microbes.

overall_design: The control and 24 h treatment groups were fed, respectively, with sucrose and bacterial solution. After 18 h, the 6 h treatment group was fed with bacteria. At 24 hours post-infection (hpi), approximately 1,000 whiteflies were collected from the control, 6 and 24 hpi treatments, respectively. Total RNA was purified with SV total RNA isolation kit (Promega). The libraries were sequenced by using Illumina HiSeq 2000 platform at the Beijing Genomics Institute (Shenzhen, China). Each channel generates 6 million reads.

first: Chang-Rong

last: Zhang

first: Shan

first: Shu-Sheng

last: Liu

first: Xiao-Wei


title: Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans.

status: Public on Dec 01 2014

city: Bern

first: Peter

last: Meister

state: Schweiz

address: Baltzerstrasse 4

department: Institute of Cell Biology

country: Switzerland

institute: University of Bern

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The essential adaptation of X-linked gene expression to the X chromosome copy number variation (called dosage compensation, DC) has been widely studied as a model of chromosome-wide gene regulation. In C. elegans, DC is achieved by two fold downregulation of gene expression from both X copies in hermaphrodites. The dosage compensation complex (DCC), a multiprotein complex structurally similar to mitotic condensins, restricts RNA polymerase progression. Higher order chromatin structures have therefore long been suggested to regulate X-linked gene expression. Here we show that in C. elegans males, the single X chromosome interacts broadly with nuclear pores, while in hermaphrodites the DCC impairs this interaction. Using microscopic measurements we find that the X chromosome is located at the nuclear periphery in males and internal in hermaphrodites, while compaction was higher for the X chromosome than for autosomes but surprisingly comparable between sexes. Mechanistically, we show that a single motif enriched on X, sufficient for DCC loading in hermaphrodites, autonomously targets an autosomal locus to the nuclear periphery specifically in males. Using DNA adenine methyltransferase identification (DamID), we demonstrate that the perinuclear interaction domains are nuclear pores. Dynamic polymer modeling shows that this discrete pore anchoring can explain the high compaction of the male X chromosome. Together, our results put forward a structural model of DC, by demonstrating that X-specific sequences mediate interactions with nuclear pores, thereby locating the X chromosome in active perinuclear domains, while the DCC physically moves X-linked genes away from these transcriptionally active neighborhoods.

overall_design: Examination of DamID pattern in 3 different strains with at least 2 replicates per strain, in male or hermaphrodite L4 animals

first: Rahul

last: Sharma

first: Daniel

last: Jost

first: Jop

last: Kind

first: Georgina

last: Gómez-Saldivar

first: Bas

last: van Steensel

last: Askjaer

first: Cédric

last: Vaillant


title: Dynamics and Characterization of Small RNAs of Virus and Host Origin from Wheat Streak Mosaic Virus- and/or Triticum Mosaic Virus-infected Susceptible and Temperature-Sensitive Resistant Wheat Cultivars

city: Lincoln

first: Jean-Jack

middle: M.

last: Riethoven

state: NE

address: 1901 Vine Street

department: Beadle Center for Biotechnology

country: USA

institute: University of Nebraska-Lincoln

type: Non-coding RNA profiling by high throughput sequencing

summary: Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are type members of Tritimovirus and Poacevirus genera, respectively, in the family Potyviridae, and are transmitted by wheat curl mites.  Co-infection of these two viruses causes synergistic interaction with increased virus accumulation and disease severity in wheat. In this study, we examined the effects of synergistic interaction between WSMV and TriMV on endogenous small (s) RNAs and virus-specific small interfering RNAs (vsiRNAs) in susceptible (Arapahoe) and temperature-sensitive resistant (Mace) wheat cultivars at 27ºC and 18ºC. Single- and double-infections in wheat caused a shift in the profile of endogenous sRNAs from 24 nt being the most predominant in healthy plants to 21 nt in infected wheat. Additionally, we report high-resolution vsiRNA maps of WSMV and TriMV in singly- and doubly-infected wheat cultivars Arapahoe and Mace at 18ºC and 27ºC. Massive amounts of 21 and 22 nt vsiRNA reads were accumulated in Arapahoe at both temperatures and in Mace at 27ºC but not at 18ºC. The plus- and minus-sense vsiRNAs were distributed throughout the genomic RNAs in Arapahoe at both temperature regimens and in Mace at 27ºC, although some regions of genomic RNAs serve as hot-spots with an excessive number of vsiRNAs. The positions of vsiRNA peaks were conserved among wheat cultivars Arapahoe and Mace, suggesting that Dicer-like enzymes of susceptible and resistant wheat cultivars are similarly accessed the genomic RNAs of WSMV and TriMV.  Additionally, several cold-spot regions were found in the genomes of TriMV and WSMV with no or a few vsiRNAs, indicating that certain regions of WSMV and TriMV genomes are not accessible to Dicer-like enzymes. The high-resolution map of endogenous and vsiRNAs from wheat cultivars synergistically infected with WSMV and TriMV at two temperature regimens form a foundation for understanding the virus-host interactions, effect of synergistic interactions on host defense mechanisms, and virus resistance mechanisms in wheat.

overall_design: Small RNA was sequenced from two wheat cultivars (Mace and Araphahoe), at two temperatures 18ºC and 27ºC, for healthy (control/uninfected), infected with wheat streak mosaic virus (WSMV), infected with Triticum mosaic virus (TriMV), and a double-infecttion of WSMV and TriMV.

first: Satyanarayana

last: Tatineni

first: Amitava

last: Mitra

first: Robert

last: Graybosch

first: Roy

last: French


title: Assessing inter- and intra-individual DNA methylation profiles in human brain samples

city: Edinburgh

first: Rob

middle: S

last: Illingworth

state: Midlothian

address: Centre for Regenerative Medicine

department: Centre for regenerative Medicine

country: United Kingdom

institute: The University of Edinburgh

type: Methylation profiling by high throughput sequencing

summary: We report the application of single molecule-based sequencing technology in combination with MBD affinity purifcation (MAP-seq) to generate methylation maps in a panel of human brain samples. Here we assay the methylation status of the human genome in eight distinct brain regions from three individuals in addition to a further thirty six hippocampal samples. The cerebellum was a consistent outlier compared to all other regions, showing over 16,000 differentially methylated regions (DMRs). Unexpectedly, the sequence properties of hypo- and hyper- methylated domains in cerebellum were distinct. In contrast we found very few DMRs that could distinguish between regions of the cortex, limbic system and brain stem. Inter-individual DMRs were readily detectable, however with the exception of cerebellum, DNA methylation patterns are more similar between different brain regions from the same individual, than they are for a single brain region between different individuals.

overall_design: Examination of DNA methylation status in human brain samples.

first: Ulrike

last: Gruenewald-Schneider

first: Dina

last: DeSousa

first: Shaun

last: Webb

first: Cara

last: Merusi

first: Alastair

middle: R

last: Kerr

first: Keith

middle: D

last: James

first: Colin

last: Smith

last: Walker

middle: M

last: Andrews

first: Adrian

middle: P

last: Bird


title: Genome-wide analysis of transcriptome and translatome following eIF4A1 knockdown in MCF7 cells [RNA-Seq]

city: Cambridge

first: Roslin

last: Russell

address: Robinson Way

institute: Cambridge Research Institute, Cancer Research UK

type: Expression profiling by high throughput sequencing

summary: To identify which genes were regulated by mRNA helicase activity, the effect of eIF4A1 knockdown on the MCF7 cell transcriptome and translatome was determined. eIF4A1-dependent mRNAs were highly enriched for several classes of genes with oncogenic potential, which leads to a model whereby dysregulation of mRNA unwinding contribues to the malignant phenotype in breast cancer cells via preferential translation of a subset of genes.

overall_design: Total, subpolysomal and polysomal RNA was isolated from MCF7 cells treated with either control siRNAs or siRNAs directed against eIF4A1 (48h post-transfection).

first: Angelika

last: Modelska


title: Coffea canephora sRNA profiling

city: Paterna

first: Regis

middle: Lopes

last: Correa

state: Valencia

address: CL. Catedrático Agustín Escardino 9

country: Spain

summary: SRNAs from field collected Coffea canephora leaves were profiled by Illumina sequencing and 63 unique microRNA genes belonging to 34 families were found.

overall_design: Leaves from 5 field collected plants were pooled for RNA extraction and sRNA sequencing.

first: Daniela

last: Ferreira

first: Guilherme

last: Loss-Morais

first: Rogério

last: Margis

first: Márcio

last: Alves-Ferreira


title: The Estrogen receptor alpha regulated NEAT1 long non-coding RNA promotes prostate cancer progression

city: NEW YORK

first: Dimple

last: Chakravarty

state: NY

address: Belfer Research Building 413 E 69 St., New York

department: Pathology

institute: Weill Cornell Medical College

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: The Estrogen receptor alpha regulated NEAT1 long non-coding RNA promotes prostate cancer progression [RNA-Seq]

summary:
Prostate glands predominantly exhibit androgen dependence, but increasing evidence suggests that estrogen receptor signaling is involved in its development and pathogenesis. By integrating ChIP sequencing for estrogen receptor alpha (ERα) with transcriptome sequencing data from prostate cancer samples, we found ERα to significantly influence the noncoding transcriptome in prostate cancer. We identified one such long noncoding RNA, NEAT1, to play an important role in prostate cancer progression through direct regulation of transcription of its target genes. NEAT1, in an ERα dependent manner, promotes prostate tumorigenesis by interacting with and modulating chromatin state at promoters of prostate cancer specific signature genes. NEAT1 expression is positively correlated with PSMA in prostate adenocarcinoma and with B3GAT1 in neuroendocrine prostate cancer. This study identifies NEAT1 as a novel biomarker or therapeutic target in prostate cancer and also suggests that co-targeting ERα and androgen receptor (AR) may be effective for a subset of patients with advanced prostate cancer and with NEAT1 overexpression.
mRNA profiles of MEF cell lines prepared from E13.5 embryos of wild-type (WT) and NEAT1 knockout (KO; NEAT1−/−) mice  were generated by deep sequencing, using Illumina HiSeq 2000. Strand specific mRNA profiles of VCaP and VCaP ERa cell lines were generated by deep sequencing, using Illumina GA IIx.

overall_design: RNA sequencing of MEF (WT vs NEAT1 KO) and VCaP (control vs ERa)

first: Andrea

last: Sboner

first: Mark

middle: A

last: Rubin


title: The Estrogen receptor alpha regulated NEAT1 long non-coding RNA promotes prostate cancer progression [ChIP-Seq]

summary: Prostate glands predominantly exhibit androgen dependence, but increasing evidence suggests that estrogen receptor signaling is involved in its development and pathogenesis. By integrating ChIP sequencing for estrogen receptor alpha (ERα) with transcriptome sequencing data from prostate cancer samples, we found ERα to significantly influence the noncoding transcriptome in prostate cancer. We identified one such long noncoding  RNA, NEAT1, to play an important role in prostate cancer progression through direct regulation of transcription of its target genes. NEAT1, in an ERα dependent manner, promotes prostate tumorigenesis by interacting with and modulating chromatin state at promoters of prostate cancer specific signature genes. NEAT1 expression is positively correlated with PSMA in prostate adenocarcinoma and with B3GAT1 in neuroendocrine prostate cancer. This study identifies NEAT1 as a novel biomarker or therapeutic target in prostate cancer and also suggests that co-targeting ERα and androgen receptor (AR) may be effective for a subset of patients with advanced prostate cancer and with NEAT1 overexpression.

overall_design: ERa ChIP seq in 4 different cell lines


title: DNA methylation analysis of human atherosclerotic plaques in comparison to healthy mammary arteries

city: Kuopio

first: Einari

last: Aavik

address: Neulaniementie 2

department: A.I. Virtanen Institute

country: Finland

institute: University of Eastern Finland

summary:
We report differential DNA methylation results from human femoral atherosclerotic plaques compared to healthy mammary arteries. 5-methylcytosine containing DNA fragments are captured by methylcytosine binding domain - based MethylCapture assay. Captured fragments are sequenced using Illumina paired-end approach and mapped to human genome (hg19).
Results. Hypomethylation of chromosomal DNA predominates in atherosclerotic plaques. Chr14q32.2 was identified for the first time as an extensively hypomethylated area in atherosclerosis with highly induced expression of miR127, -136, -410, -431, and -432

overall_design: DNA methylation in 8 human atherosclerotic plaques was compared to 5 healthy mammary arteries.

first: Olli

last: Leppänen

first: Jan-Hinrich

last: Bräsen

first: Ulrich

last: Beschorner

first: Thomas

last: Zeller

first: Maarten

last: Braspenning

first: Wim

last: van Criekinge

first: Kimmo

last: Mäkinen

first: Seppo

last: Ylä-Herttuala


title: Elucidation of stress memory inheritance through epigenome alterations in Brassica rapa plants [small RNA-seq]

city: Lethbridge

first: Igor

last: Kovalchuk

address: 4401 University Drive

country: Canada

institute: UofL

summary: In the current study we have profiled changes in the smRNome of somatic and reproductive tissues of B.rapa plants following heat shock stress exposure. For this we used Illumina GAIIx platform. We have demonstrated that the highest tissue-specific alterations in the smRNome profile are detected in tissues that were not directly exposed to stress, namely, in the endosperm and pollen. The progeny of exposed plants also exhibited significant alterations in the smRNA as compared to progeny of control plants.

overall_design: The smRNome profile was assessed after heat shock treatment in the following tissues of Brassica rapa plants: parental leaves, inflorescence meristem, pollen, unfertilized ovules, 24-hour post-fertilization ovules, embryo, endosperm and leaf tissues of progeny plants.

first: Bilichak

last: Andriy

first: Kovalchuk

last: Igor


title: High-throughput sequencing of HMW RNAs (4-10 kb) from Drosophila Dnmt2 mutants during heat shock recovery

city: Vienna

first: Schaefer

last: Matthias

state: AT

address: Schwarzspanierstrasse 17-I

department: Cell and Developmental Biology

country: Austria

institute: Medical University Vienna, Center for Anatomy & Cell Biology

summary: Dnmt2 genes are highly conserved tRNA methyltransferases with biological roles in cellular stress responses. Dnmt2 has recently been implicated in transposon silencing in Drosophila but the exact molecular mechanisms are unclear. Adult Dnmt2 mutants were heat shocked and RNA sequencing was performed on visible high-molecular weight RNAs to determine the identity of up-regulated transposons. Dnmt2 mutants accumulated almost all families of transposons after heat shock, indicating a general mis-regulation of transposon silencing in Dnmt2 mutants during the stress response.

overall_design: one sample, excised, electroeluted and pooled RNA of different molecular weight, Dnmt2 mutant during recovery from a single heat shock

first: Zeljko

last: Durdevic

first: Matthias

last: Schaefer


title: Analysis of pre-mRNA splicing trans-regulation in human lymphoblastoid cell lines

city: Basel

first: Julien

last: Roux

address: Hebelstrasse 20

department: Department of Biomedicine

institute: University of Basel

summary: To understand the role of trans factors in the regulation of splicing and alternative splicing, we knocked-down 22 RNA-binding proteins previously shown or suspected to be involved in the regulation of splicing (“splicing factors”). We performed knockdown experiments in triplicate in the Hapmap lymphoblastoid cell line GM19238 using small interfering RNAs (siRNAs). RNA was extracted 72 hours after knockdown for RNA-seq library preparation. The following splicing factors were analyzed: ACIN1, ADAR, HNRPA2B1, HNRPF, HNRPH1, HNRPH2, HNRPK, HNRPL, HNRPR, PCBP2, PTBP1, RBM23, RBM39, RBMX, RNPS1, SRSF1, SRSF3, SRSF4, SRSF8, SRSF9, SRSF10 and SYNCRIP. Seven controls were generated with non-specific siRNA probes transfection. RNA-seq libraries were multiplexed and sequenced on 3 different flow-cells on an Illumina HiSeq 2000 sequencer (single-end 107 bp long reads).

overall_design: 73 samples, including 3 replicates for each of the 22 knockdowns and 7 controls

first: Bryan

last: Pavlovic

first: Jonathan

middle: K

last: Pritchard

first: Yoav

last: Gilad


title: Prevention and cure of rotavirus infection via TLR5/NLRC4-mediated production of IL-22 and IL-18

status: Public on Nov 30 2014

city: Atlanta

first: Benoit

last: Chassaing

state: GA

address: 100 Piedmont Av SE

department: Biology

institute: Georgia State University

summary: Activators of innate immunity may have potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin (IL)-22, which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection, and thus holds promise as a broad-spectrum antiviral agent.

overall_design: Total mRNA from intestinal epithelial cells of Rag1-/- mice treated with PBS, IL-18, IL-22 or IL-22/IL-18 was assayed for RNA sequencing.

first: Benyue

last: Zhang

first: Andrew

middle: T

last: Gewirtz


title: The transcriptomic response of Acinetobacter baumannii to colistin and doripenem alone and in combination in an in vitro pharmacokinetics/pharmacodynamics model

city: Clayton

first: David

middle: Richard

last: Powell

state: VIC

address: Wellington Road

department: Bioinformatics Platform

country: Australia

institute: Monash University

summary: Objectives: Colistin remains a last-line treatment for multidrug-resistant Acinetobacter baumannii and combined use of colistin and carbapenems has shown synergistic effects against multidrug-resistant strains. In order to understand the bacterial responses to these antibiotics we analysed the transcriptome of A. baumannii following exposure to each.

sample_organism: Acinetobacter baumannii ATCC 19606 = CIP 70.34 = JCM 6841

overall_design: Methods: RNA sequencing was employed to determine changes in the transcriptome following treatment with colistin and doripenem, both alone and in combination, using an in vitro pharmacokinetics/pharmacodynamics (PK/PD) model to mimic the pharmacokinetics of both antibiotics in patients.

first: John

last: Boyce


title: Pwp1 Is Required for the Differentiation Potential of Mouse Embryonic Stem Cells through Regulating Stat3 Signaling

city: Shanghai

first: Weisheng

last: Zheng

address: Siping Road, No.1239

department: Department of Bioinformatics

country: China

institute: Tongji University

summary: We show that periodic tryptophan protein 1(Pwp1), a WD-40 repeat-containing protein associated with histone H4 modification, is required for the exit of mES cells from the pluripotent state into all lineages. Knockdown of Pwp1 does not affect mES cell proliferation, self-renewal or apoptosis. However, knockdown of Pwp1 impairs the differentiation potential of mES cells both in vitro and in vivo. The PWP1 ChIP-seq results revealed that the PWP1-occupied regions were marked with significant levels of H4K20me3. Moreover, Pwp1 and H4K20me3 bind to the same sites in the upstream region of Stat3. Knockdown of Pwp1 decreases the binding of H4K20me3 to the upstream region of Stat3 gene and upregulates the expression of Stat3. Furthermore, Pwp1 knockdown(KD) mES cells recover their differentiation potential through suppressing the expression of Stat3 or inhibiting the tyrosine phosphorylation of STAT3. Together, our results suggest that Pwp1 plays important roles in the differentiation potential of mES cells.

overall_design: Examination of Pwp1 binding sites in  mouse embryonic stem cells

first: Junwei

last: Shen

first: Cizong

last: Jiang


title: High resolution chromatin dynamics during a yeast stress response

status: Public on Nov 29 2014

city: Jerusalem

first: Alon

last: Appleboim

address: Hebrew University

department: Life Sciences

country: Israel

institute: Hebrew University of Jerusalem

summary: Covalent histone modifications are highly conserved and play multiple roles in eukaryotic transcription regulation. Because of extensive crosstalk between transcriptional processes and histone modifications, steady state observations are insufficient to fully disentangle histone modification networks. As transient perturbation of feedback mechanisms can reveal network structure, we mapped 26 histone modifications genome-wide over a time course following dramatic transcriptional reprogramming – response to diamide stress in yeast. Interestingly, while we observed limited combinatorial complexity in steady-state histone modification patterns, consistent with previous studies, the combinatorial complexity of histone modification space increased modestly during the stress response, resulting from roughly 3% of nucleosomes transiently populating rare histone modification states. We show that most of these short-lived histone mark combinations result from differences in histone modification dynamics that transiently uncouple highly correlated marks, with slow histone methylation changes often lagging the more rapid changes in acetylation. Together, our results provide the first detailed view of an epigenome in transition.

overall_design: Mnase-ChIP-Seq with 26 antibodies, throuh a diamide stress response (0,4,8,15,30,60 minutes). Performed in batches, in each batch the MNase input is also sequenced in each time point.

first: Assaf

last: Weiner

first: Tsung-Han

last: Hsieh

first: Hsiuyi

middle: V

last: Chen

first: Ayelet

last: Rahat

first: Ido

last: Amit

first: Oliver

middle: J

last: Rando

first: Nir

last: Friedman


title: Circulating miRNAs of acetaminophen-overdosed patients

status: Public on Nov 28 2014

city: Maastricht

first: Julian

last: Krauskopf

address: Universiteitssingel 50

country: Netherlands

institute: Maastricht University

summary: Drug-induced liver injury (DILI) is a leading cause of acute liver failure and the major reason for withdrawal of drugs from the market. Preclinical evaluation of drug candidates has failed to detect about 40% of potentially hepatotoxic compounds in humans. At the onset of liver injury in humans, currently used biomarkers have difficulty differentiating severe DILI from mild, and/or predict the outcome of injury for individual subjects. Therefore, new biomarker approaches for predicting and diagnosing DILI in humans are urgently needed. Recently, circulating microRNAs (miRNAs) such as miR-122 and miR-192 have emerged as promising biomarkers of liver injury in preclinical species and in DILI patients. In this study, we focused on examining global circulating miRNA profiles in serum samples from subjects with liver injury caused by accidental acetaminophen (APAP) overdose. Upon applying next generation high-throughput sequencing of small RNA libraries, we identified 36 miRNAs, including 3 novel miRNA-like small nuclear RNAs, which were enriched in the serum of APAP overdosed subjects. The set comprised miRNAs that are functionally associated with liver-specific biological processes and relevant to APAP toxic mechanisms. Although more patients need to be investigated, our study suggests that profiles of circulating miRNAs in human serum might provide additional biomarker candidates and possibly mechanistic information relevant to liver injury.

overall_design: small RNA profiles of serum form 6 acetaminphen overdosed subjects and 6 controls

first: Jos

last: Kleinjans


title: iCD8a cells study

status: Public on Nov 27 2014

city: Nashville

first: Yan

last: Guo

state: TN

address: 639 Preston Research Bld

institute: Vanderbilt Cancer Center

summary: Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8a cells, characterized by expression of CD8a homodimers. iCD8a cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8a cells depends on expression of interleukin-2 receptor g chain (IL-2Rgc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8a cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8a cells in immune responses associated with the intestinal epithelium.

overall_design: Three different populations of immune cells present in the intestine epithelium of RAG-2 deficient mice were compared at the transcriptome level. These populations were defined as: a. CD45+CD8alpha+ (iCD8a cells), b. NKp46+NK1.1+ (group 1 ILC), and c.CD45+CD8alpha-CD11b+ (myeloid cells).  High quality RNA was sequenced at  the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core using an Illumina HiSeq 2500. RAN data alignment was performed by top hat, followed by gene quantification (FPKM) using Cufflinks. Additional read count was generated using HTSeq. Differential expression analysis was carried out using both FPKM and read count-based methods. Pathway and network analyses were performed using Ingenuity.

first: Danyvid

last: Olivares-Villagomez

first: Luc

last: Van Kaer


title: Transcriptome analysis of Jurkat T cells expressing MALT1 or its mutants MALT1-R149A and MALT1-C464A or the MALT1-R149A-C464A double mutant.

city: Leuven

first: Thijs

last: Baens

address: Herestraat 49

country: Belgium

institute: VIB

summary:
Purpose: study the role of MALT1 auto-proteolysis in T cell receptor mediated activation of NF-kB.
Methods: Jurkat cells were generated that express wild type MALT1, the auto-cleavage deficient MALT1-R149A mutant, the catalytic inactive MALT1-C464A mutant or the R149A-C464A double mutant (RACA). Expression of endogenous MALT1 was inactivated using TALEN technology for the Jurkat cells expressing MALT1-R149A (JDM-RA) and MALT1-C464A (JDM-CA). Illumina HISeq 2000 deep sequencing was performed to determine the mRNA profiles for MALT1, JDM-RA, JDM-CA and RACA cells in unstimulated conditions or after treatment with 75ng/ml PMA and 150 ng/ml ionomycin for 3 or 18 hrs.
Results: PMA ionomycin stimulation of the MALT1 auto-cleavage defective JDM-RA cells fails to activate NF-kB-dependent transcription like for the MALT1 catalytic inactive JDM-CA cells and the double RACA mutant cells.
Conclusion: MALT1 autoproteolysis is essential for transcription of NF-kB target genes

overall_design: mRNA profiles of Jurkat expressing MALT1, MALT1-R149A, MALT1-C464A and MALT1-R149A-C464A after 0, 3 and 18 hours of stimulation with PMA and Ionomycin were generated by deep sequencing, in duplicate, using  Illumina HISeq 2000

first: Mathijs

first: Peter

last: Marynen


title: RNF17 blocks promiscuous activity of PIWI proteins in mouse testes [5'RACE]

status: Public on Dec 17 2014

city: Cold Spring Harbor

first: Vasily

last: Vagin

address: 1 Bungtown Rd

institute: Cold Spring Harbor

summary: PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction.

overall_design: Examination of 5'RACE in heterozygous and homozygous RNF17 adult testes or MIWI/MILI immunoprecipitates

first: Kaja

last: Wasik

last: Tam

first: Yuan

last: Hao

first: Ilaria

last: Falciatori

first: Molly

last: Hammell

first: Gregory

last: Hannon


title: Whole genome microRNA expression profiling of long-lived daf-2(e1370) mutant in C. elegans at young adult stage

city: New Delhi

first: Arnab

last: Mukhopadhyay

address: Aruna Asaf Ali Marg

country: India

institute: National Institute of Immunology

summary: In order to get an insight into the complex interplay of miRNAs in insulin signaling, we profiled the small RNA polulation of daf-2(e1370) worms at young adult stage by Next Generation sequencing.

overall_design: We performed Next Generation sequencing to compare miRNA profiles of wild-type and long-lived daf-2(e1370) mutant at young adult stage

first: A

last: Pandit

first: Vaibhav

last: Jain


title: Whole genome microRNA  expression profiling during C. elegans dietary restriction

summary: In order to get an insight into the complex interplay of miRNAs in dietary restriction, we profiled the small RNA polulation of wild-type and eat-2(ad1116) worms on Day 1 and Day 8 of adulthood by Next Generation sequencing. We compared the normalized expression  of wild-type vs eat-2(ad1116) on either day 1 or day 8 and found that most of the miRNAs were distinctly upregulated in eat-2(ad1116) on day 1.

overall_design: We performed Next Generation sequencing to compare miRNA profiles of wild-type and eat-2(ad1116) at Day 1 and Day 8


title: Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases

city: Stanford

first: Richard

middle: L

last: Frock

state: California

address: 269 Campus Drive

department: Radiation Oncology

institute: Stanford University

summary: We describe a robust linear amplification-mediated high-throughput genome-wide translocation sequencing (HTGTS) method that identifies endogenous or ectopic "prey" DNA double-stranded breaks (DSBs) across the human genome based on their translocation to "bait" DSBs generated by engineered nucleases.  HTGTS with different Cas9:gRNA or TALEN-nuclease on-target baits revealed off-target hotspots for given nucleases that ranged from few or none to dozens or more, and greatly extended known off-target numbers for certain previously characterized engineered nucleases by more than 10-fold. Beyond various types of nuclease off-target collateral damage, we also identified collateral damage in the form of translocations between bona fide nuclease targets on homologous chromosomes.  Based on frequent non-specific DSBs making any given human chromosome an HTGTS hotspot region for bait DSBs within it, we found that HTGTS also reveals wide-spread, low-level DSB-generating activities of engineered nucleases.  Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-targets genome-wide and suggested other strategies to enhance desired nuclease activities.

overall_design: Examination of 9 different target loci using Illumina Miseq

first: Robin

last: Meyers

first: Frederick

middle: W

last: Alt


title: Identification of mRNAs and lincRNAs associated with lung cancer progression using next-generation RNA sequencing from laser micro-dissected archival FFPE tissue specimens

city: Cleveland

first: Xiaodong

last: Bai

address: 10900 Euclid Avenue

department: Center for RNA Molecular Biology

institute: Case Western Reserve University

summary: Adenocarcinoma in situ (AIS) is considered an intermediate step in the progression of normal lung tissue to invasive adenocarcinoma.  However, the molecular mechanisms underlying this progression remain to be fully elucidated due to difficulties in obtaining and preserving clinical samples for downstream analyses.  Formalin fixation and paraffin embedding (FFPE) is a tissue preservation system that is widely used as a means for long-term storage.  Until now, challenges in working with FFPE have precluded using new RNA sequencing technologies (RNA-seq), which would help clarify some of the key pathways affected in the transition from normal to AIS to invasive adenocarcinoma.  Recent technological advances have made it possible to sequence RNA from archival tissues.  Also, isolation techniques including laser-capture micro-dissection provide the ability to select histopathologically distinct tissues, allowing researchers to study transcriptional variations between tightly juxtaposed cell and tissue types.  Utilizing these technologies and new alignment tools we examined differential expression of long intergenic non-coding RNAs and mRNAs across normal, AIS and invasive adenocarcinoma samples from six patients to identify possible markers of lung cancer progression. RNA extracted and sequenced from these 18 samples generated an average of 198 million reads per sample.  After alignment and filtering, uniquely aligned reads represented an average 35% of the total reads.  We detected differential expression of a number of lincRNAs and mRNAs when comparing normal to AIS, or AIS to invasive adenocarcinoma.  Of these, 5 lincRNAs and 31 mRNAs were consistently up- or down-regulated from normal to AIS and more so to invasive carcinoma.  We validated the up-regulation of two mRNAs and one lincRNA by RT-qPCR as proof of principle. Our findings indicate a potential role of not only mRNAs, but also lincRNAs in invasive adenocarcinoma.  We anticipate that our current findings will lay the groundwork for future experimental studies of candidate RNAs from FFPE samples to identify their functional roles in lung cancer.

overall_design: RNA-Seq in 6 patients in norma lung, adenocarcinoma in situ of the lung, and invasive lung carcinoma

first: Matthew

last: Morton

first: Rom

last: Leidner

first: Cheryl

last: Thompson


title: A burst of miRNA innovation in the early evolution of butterflies and moths

status: Public on Nov 26 2014

city: Oxford

first: Peter

middle: W. H.

last: Holland

address: South Parks Road

department: Department of Zoology

institute: University of Oxford

summary: MicroRNAs (miRNAs) are involved in post-transcriptional regulation of gene expression. Since several miRNAs are known to affect the stability or translation of developmental regulatory genes, the origin of novel miRNAs may have contributed to the evolution of developmental processes and morphology. Lepidoptera (butterflies and moths) is a species-rich clade with a well-established phylogeny and abundant genomic resources, thereby representing an ideal system in which to study miRNA evolution. We sequenced small RNA libraries from developmental stages of two divergent lepidopterans, Cameraria ohridella (Horse chestnut Leafminer) and Pararge aegeria (Speckled Wood butterfly), discovering 90 and 81 conserved miRNAs respectively, and many species-specific miRNA sequences. Mapping miRNAs onto the lepidopteran phylogeny reveals rapid miRNA turnover and an episode of miRNA fixation early in lepidopteran evolution, implying that miRNA acquisition accompanied the early radiation of the Lepidoptera. One lepidopteran-specific miRNA gene, miR-2768, is located within an intron of the homeobox gene invected, involved in insect segmental and wing patterning. We identified cubitus interruptus (ci) as a likely direct target of miR-2768, and validated this suppression using a luciferase assay system. We propose a model by which miR-2768 modulates expression of ci in the segmentation pathway and in patterning of lepidopteran wing primordia.

sample_organism:
Pararge aegeria
Cameraria ohridella

platform_organism:
Pararge aegeria
Cameraria ohridella

overall_design: Examination of the small RNA complements pooled across life cycle stages in each of Cameraria ohridella and Pararge aegeria.

first: Shan

last: Quah

first: Jerome

middle: H

last: Hui


title: Expression profiling, Miz1 location analysis and histone modification analysis in 70Z/3 and P6D4 cells

city: Montreal

first: Möröy

last: Tarik

state: Quebec

address: 110 Pine avenue West

institute: Insititut de Recherches Cliniques

summary: Expression profiling by RNA-Seq was performed on 70Z3 and P6D4 cells as well as ChIP-Seq location analysis of Miz1, H3K4Me3, H3K27Ac and H3K9Ac. This was done as part of a larger study regarding the role of Miz1 as a regulator of Trp53 translation.

overall_design: RNA-Seq was carried out in triplicate for each cell line. Miz1 and histone mark ChIP-Seqs were carried out once for each cell line from a single cell lysate preparation and thus have the same input control.

first: Marissa

last: Rashkovan

first: Tarik

last: Möröy

first: Charles

last: Vadnais


title: Characterization of a network of tumor suppressor microRNA's in T Cell acute lymphoblastic leukemia

city: New York

first: Hans-Guido

last: Wendel

address: 1275 York Avenue

institute: Sloan Kettering Cancer Center

summary:
Purpose: The purpose of this study is to identify functionally inter-connected group of miRNAs whose reduced expression promotes leukemia development in vivo. We searched for relevant target genes of these miRNAs that are upregulated in T-ALL relative to controls.
Methods: In order to examine the global gene expression, we generated 9 T-ALL patients and 4 normal controls by deep sequencing using Illumina Hi-Seq sequencer. The sequence reads that passed quality filters were analyzed using Spliced Transcripts Alignment to a Reference aligner (STAR) followed by differential gene expression analysis using DESeq.
Results: Using an optimized data analysis workflow, we mapped reads per sample to the human genome (build hg19) and identified transcripts in both patient and controls with STAR workflow. We applied a machine learning approach to eliminate targets with redundant miRNA-mediated control. This strategy finds a convergence on the Myb oncogene and less prominent effects on the Hpb1 transcription factor. The abundance of both genes is increased in T-ALL and each can promote T-ALL in vivo.
Conclusion: Our study reveals a Myc regulated network of tumor suppressor miRNAs in T-ALL. We identified a small number of functionally validated tumor suppressor miRNAs.  These miRNAs are repressed upon Myc activation and this links their expression directly to Myb a key oncogenic driver in T-ALL.

overall_design: Examination of global gene expression in 9 T-ALL patients and 4 normal controls using total RNA sequencing. BaseMeanA in DESeq_results.xlsx is the control.

first: Viraj

last: Sanghvi


title: Epigenetic inheritance uncoupled from sequence-specific recruitment

city: Boston

first: Gloria

last: Jih

state: MA

address: 240 Longwood Ave, LHRRB 517

department: Cell Biology

institute: Harvard Medical School

summary: We used ChIP-seq to examine the requirements for inheritance of H3K9me at endogenous and ectopic loci.

overall_design: H3K9me2 and H3K9me3 ChIP-seq in multiple S. pombe strains

first: Danesh

last: Moazed


title: JBP1-seq: a fast and efficient method for genome-wide profiling of 5hmC

city: Irvine

first: Xueguang

last: Sun

state: CA

address: 17062 Murphy Ave

institute: zymo research corp

summary: We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines  the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and nextgeneration  sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC).  Compared with the original JBP1, this new recombinant JBP1 was biotinylatedin vivo and conjugated  to magnetic beads via biotin-streptavidin interactions. These modifications allowed a more efficient  and consistent pull-down of β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC), and sequence-ready  libraries can be generated within 4.5 hours from DNA inputs as low as 50 ng. 5hmC enrichment of  human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly  higher than the 4,003 peaks enriched using the old JBP1. Comparison of the technical duplicates and  validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq  provides a fast, efficient, cost-effective method for accurate 5hmC genome-wide profiling.

overall_design: An improvement of JBP1-Seq

first: Libin

last: Cui

first: Tzu

last: Chung

first: Darany

last: Tan

first: Xi-Yu

last: Jia


title: KdmA, a histone H3 demethylase with bipartite function, differentially regulates primary and secondary in Aspergillus nidulans metabolism

city: Tulln

first: Harald

last: Berger

state: NOE

address: Konrad-Lorenzstraße 43

department: DAGZ

institute: Universität f. Bodenkultur Wien

summary: A. nidulans kdmA encodes a member of the KDM4 family of jumonji histone demethylase proteins, highly similar to metazoan orthologues both within functional domains and in domain architecture. This family of proteins exhibits demethylase activity toward lysines 9 and 36 of histone H3 and plays a prominent role in gene expression and chromosome structure in many species. Mass spectrometry mapping of A. nidulans histones revealed that around 3% of bulk histone H3 carried trimethylated H3K9 (H3K9me3) but more than 90% of histones carried either H3K36me2 or H3K36me3. KdmA functions as H3K36me3 demethylase and has roles in transcriptional regulation. Genetic manipulation of KdmA levels is tolerated without obvious effect in most conditions, but strong phenotypes are evident under various conditions of stress. Transcriptome analysis revealed that – in submerged early and late cultures – between 25% and 30% of the genome is under KdmA influence, respectively. Transcriptional imbalance in the kdmA deletion mutant may contribute to the lethal phenotype observed upon exposure of mutant cells to low-density visible light on solid medium. While KdmA acts as transcriptional co-repressor of primary metabolism (PM) genes it is required for full expression of several genes involved in biosynthesis of secondary metabolites (SM).

overall_design: Two strains, wild type and kdmA deletion, at two conditions, growth at primary (17h) and secondary (48h), were analyzed. Each sample was replicated.

first: Agnieszka

last: Gacek

first: Luke

last: Noble

first: Clemens

last: Gruber

first: Ana

last: Marcos

first: Michael

last: Sulyok

first: Joseph

last: Strauss

first: Alex

last: Andrianopoulos


title: Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer [ChIP-seq]

city: Cambridge

first: Chandra

last: Chilamakuri

address: Robinson Way

institute: Cancer Research UK Cambridge Institute

summary: Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels.  HIF1A is a major regulator of this process but activation of HIF1A under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of Hypoxia Inducible Factor 1A (HIF1A) transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.

overall_design: In an attempt to identify the ARRB1 cistrome in prostate cancer cells, C4-2 prostate cancer cells expressing endogenous levels of ARRB1 were used to ChIP for ARRB1, p300 (previously shown to interact with ARRB1within transcriptional complexes), RNA PolII and histone markers H3K4me1 and H4K4me3 (markers for enhancer and promoter regions, respectively). Cells were untreated and cultured in FBS supplemented with 10%FBS. In parallel, C4-2 cells stably expressing a nuclear form of ARRB1 (nucARRB1) were also used to ChIP the same complexes under the same conditions. Finally, human prostate tissue was used to ChIP for ARRB1 and histone markers.

first: Vincent

last: Zecchini

first: Rory

last: Stark

first: Roslin

last: Russell


title: Pre-mRNA Splicing is Facilitated by an Optimal RNA Polymerase II Elongation Rate

status: Public on Nov 25 2014

city: Aurora

first: David

middle: L.

last: Bentley

state: CO

department: Biochemistry and Mol. Genetics

institute: U. Colorado School of Medicine

summary: Transcription and pre-mRNA alternative splicing was analyzed by  in isogenic human HEK293 cell lines that inducibly express a-amanitin resistant mutants of the RNA polymerase II large subunit with slow and fast elongation rates.

overall_design: Alternative splicing was analyzed by, RNA-seq and RASL-seq of polyA+ RNA from a-amanitin treated cells; transcription elongation rate was analyzed by BrUTP-labelled GRO-seq of a-amanitin treated cells at time points after release from a DRB (5,6-dichloro-1-bold beta-D-ribofuranosylbenzimidazole) block.

first: Hyunmin

last: Kim

first: Xiang-Dong

last: Fu


title: ARRB1 regulates prostate cancer cell metabolism

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

overall_design: Refer to individual Series


title: The SNF2 and RING domain proteins FRG1 and 2 are required for RNA-directed DNA methylation in Arabidopsis [mRNA-Seq]

city: Dallas

first: Hume

last: Stroud

state: TX

address: 5323 Harry Hines Blvd

institute: UT Southwestern

summary: DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: MET1, CMT3, DRM2, and CMT2. However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as DICER-LIKE 2, 3 and 4, and IDN2- LIKE 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, since the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-RING-HELICASE-LIKE1 and 2 (FRG1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRG1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRG1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as novel components of the RdDM machinery.

overall_design: mRNA profiles of 14 days old seedlings of Columbia 0 wild type and frg1-1 frg2-1 double mutants, 3 biological replicates each.

first: Martin

last: Groth

first: Steve

middle: E

last: Jacobsen


title: The SNF2 and RING domain proteins FRG1 and 2 are required for RNA-directed DNA methylation in Arabidopsis


title: Transcriptome profiling of a mouse model of alveolar soft part sarcoma

city: Salt Lake City

first: Kevin

middle: B

last: Jones

state: Utah

address: 2000 Circle of Hope Drive, Room 4263

department: Department of Orthopaedics

institute: University of Utah

summary: In order to dtermine how well a mouse genetic model of alveolar soft part sarcoma (ASPS) mimics the human disease, five human ASPS tumor samples and three normal skeletal muscle samples were profiled by RNAseq and compared to samples from five mouse tumors induced by expression of ASPSCR1-TFE3 and three normal mouse skeletal muscle samples, also profiled by RNAseq.

overall_design: The reference was really comparing 5 human ASPS tumors to 5 mouse tumors that histologically mimic ASPS, but using skeletal muscle controls (3 from each species) as a sounding board for differential expression.


title: Assessment of patient-derived tumour xenografts (PDXs) as a discovery tool for cancer epigenomics

city: Toronto

first: Paul

last: Guilhamon

state: ON

address: 686 Bay Street

institute: SickKids

type: Methylation profiling by genome tiling array


title: The SUVR2 associated proteins FRINGE1 and 2 are required for RdDM in Arabidopsis [Bisulfite-seq]

summary: DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes MET1, CMT3, DRM2 and CMT2. However, new methylation patterns are established exclusively by the enzyme DRM2, which acts within a pathway termed RNA-directed DNA methylation (RdDM).  Some RdDM components belong to gene families and have partially redundant functions (i.e. DICER LIKE 2, 3 and 4 and IDN2 PARALOG 1 and 2). Traditional screens, which usually affect single genes, usually fail to detect genes if they are redundant, since the loss of one gene is compensated by a related gene. In an effort to circumvent this caveat, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. We report the discovery of two redundant proteins, SNF2-RING-HELICASE1 and 2 (FRINGE1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRINGE1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRINGE1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo.  Combined our results identify FRINGE1 and FRINGE2 as novel components of the RdDM machinery.

overall_design: One fringe 1/2 sample analyzed.


title: Phenazine regulated genes in Pseudomonas chlororaphis 30-84

city: Vernon Hills

first: DONGPING

last: WANG

state: IL

address: 777 Forest Edge Dr

department: Amlan

institute: OilDri Corporation of America

summary: Pseudomonas chlororaphis strain 30-84 is an effective biological control agent against take-all disease of wheat. Phenazines, bacterial secondary metabolites produced by 30-84, are essential for 30-84 to inhibit fungal pathogens, form biofilms, and effectively colonize the rhizosphere. However, how the bacteria themselves respond to phenazines remains unknown. In this study, we conducted an RNA-seq analysis by comparing the wild type strain with a phenazine deficient mutant. RNA-seq analysis identified over 200 genes differentially regulated by phenazines. Consistent with previous findings in Pseudomonas aeruginosa PAO1, phenazines positively contribute to the expression of their own biosynthetic genes. Moreover, phenazine regulatory genes including the phzI/phzR quorum sensing system and the rpeB response regulatory were also expressed at high levels in the presence of phenazines. Besides phenazine biosynthesis and regulatory genes, genes involved in secondary metabolism, exopoysaccharide production and iron uptake as well as amino acid transport were identified as the major components under phenazine control, including many novel genes. We have also demonstrated that mutation of the primary siderophore gene pvdA resulted in up-regulation of phenazine genes when grown in iron-limiting media. These findings implicate phenazines as signaling molecules to regulate gene expression and hence alter metabolism in P. chlororaphis strain 30-84.

sample_organism: Pseudomonas chlororaphis subsp. aureofaciens 30-84

overall_design: A total of 4 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas chlororaphis wild type strain (2 replicates);  Pseudomonas chlororaphis ZN mutant (2 replicates).

first: Dongping

last: Wang

first: Elizabeth

last: Pierson


title: Exosomes secreted by a nematode parasite transfer small RNAs to mammalian cells and regulate genes of the innate immune system [Litomosoides sigmodontis]

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

institute: Centre for Immunity, Infection and Evolution

summary: In mammalian systems, extracellular small RNAs can operate in a paracrine manner to communicate information between cells, relying on transport within vesicles. “Foreign” small RNAs derived from bacteria, plants and parasites have also been detected in mammalian body fluids, sparking interest in whether these could mediate inter-species communication. However, there is no mechanistic framework for RNA-mediated interspecies communication and the active movement of RNA via vesicles has not been shown outside of mammals. Here we demonstrate that specific microRNAs and Y RNAs are packaged into vesicles secreted by a gastrointestinal nematode, Heligmosomoides polygyrus, which naturally infects mice.

overall_design: Total RNA was extracted from the serum of mice infected with Litomosoides sigmodontis at 60 days post infection

middle: C

first: Amy

last: Buck


title: The SNF2 and RING domain proteins FRG1 and 2 are required for RNA-directed DNA methylation in Arabidopsis [small RNA-Seq]

overall_design: Small RNA profiles of Columbia 0 wild type, frg1-1 frg2-1 double, frg1 to 5 quintuple, suvr2-1, dcl3-1 and drm1-1 drm2-2 double mutants, 3 biological replicates each.


title: A computational algorithm to predict shRNA potency

status: Public on Nov 24 2014

city: Cold Spring Harbor

first: Simon

middle: Robert Vincent

last: Knott

institute: Cold Spring Harbor Laboratory

sample_organism:
Gallus gallus
Homo sapiens
Mus musculus
synthetic construct


title: shRNA Sensor Analysis of 5' U converted shRNAs reveals 3' target bias exists in addition to 5' guide bias

summary: shRNAs selected with the shERWOOD algorithm were converted to have a U at the 5' end of their guide. When endogenous 1U shRNAs were compared to artificial shRNA via the sensor algorithm, the endogenous shRNAs were found to be more efficacious.

overall_design:
Purpose: Structural studies have hinted that the 5' end of shRNA guides is engulfed in the RISC complex. It has also been reported that shRNAs with a 5' U are more efficacious than those with other 5' caps. We wished to determine whether replacement of shRNA guide 5' nucleotides with a U, regardless of the corresponding target base, would increase their efficacy.
Method: For each gene in the "druggable genome" 10 shRNAs were selected with the shERWOOD algorithm. In each case the score was assessed as if the guide had a 5' U. Sensor constructs were designed pairing 1U-guide shRNAs with their endogenous target. shRNAs were assessed for efficacy via the shRNA sensor assay (Fellmann et al. Mol Cell 2011).
Results: shRNAs with artificial 5' Us were found to be less efficacious than those with an endogenous 5' U,

middle: R


title: Sub-genomewide shRNAs constructed using an optimized selection algorithm and microRNA backbone provide stronger evidence for follow-up studies

summary: Sub-genomewide shRNA libraries were constructed using the current RNAi consortium constructs as well as using the DSIR (siRNA algoirthm) and a novel shRNA specific algorithm (shERWOOD). All libraries were placed into mir30 expression vectors. The shERWOOD libraries were also placed in a vector harboring an optimized mir cassette (ultramir). Each library was screened using the pancreatic cell line A385. A concensus set of essential genes identified as the set for which two shRNAs depleted in each of the libries. For these genes, a great percentage of shERWOOD seletected shRNA depleted. In addition the placement of shERWOOD selected constructs into ultramir scaffoled increased the rate of shRNA depletion for essential genes further.

overall_design:
Purpose: shRNA screens were carried out using various library construction strategies to identify the strategy that provides the best shRNA screening results.
Method: Libraries were constructed using the TRC shRNA set as well as shRNAs identified using the DSIR and shERWOOD algorithms.
shRNA libraries were cloned into mir30 expression vectors. shERWOOD shRNAs were also cloned into an expression vector harboring an optimized microRNA scaffold termed ultramir.
Each resultant library was screened using the pancreatic cell line A385.
Each library was analyzed separately to identify a set of genes where at least two shRNAs depleted. These gene sets were intersected to develop a set of essential genes.
Results: The shERWOOD shRNA libraries provided the highest number depleting shRNAs for each essential gene. Further these shRNAs depleted to a greater extent than did the shRNAs from the other libraries.
When shERWOOD libraries were placed into the ultramir cassette a greater number of shRNAs per essential gene depleted.


title: shRNA off target analysis via RNAseq

summary: shRNAs were assessed for off-target effects by comparing the gene expression profiles of cells that they had been infected into. shRNAs designed with the shERWOOD algorithm and house in the ultramir microRNA scafold were found to have very little off targeting.

overall_design:
Purpose: A major detriment to RNAi is off-targeting. We wished to assess the level of off targeting of microRNA (ultramiR) housed shERWOOD shRNAs as compared to similar shRNAs in the TRC collection.
Methods: 5 shRNAs targeting each of two genes were infected into the 4T1 cell line. For each gene one shRNA was selected from the TRC collection and one based on the shERWOOD algorithm. For each gene, the exrpession profiles of the corresponding shRNA infected cells were compared using RNAseq.
Conclusions:  Highly similar profiles were observed between shERWOOD selected shRNAs. TRC shRNAs produced profiles indicative of off-targeting.


title: An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments

city: Worcester

first: Job

last: Dekker

state: Massachusetts

address: 368 Plantation Street

department: Program in Systems Biology

institute: UMass Medical School

summary: Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs, variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2–3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.

overall_design: Deep sequencing libraries from either a randomized RNA oligo or an equimolar miRNA mix were analyzed for evenness of capture.

first: Erin

last: Heyer

first: Hakan

last: Ozadam

first: Emiliano

middle: P

last: Ricci

first: Can

last: Cenik

first: Melissa

last: Moore


title: Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

institute: Altos Labs


title: Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast [RNA-Seq]

summary: Reprogramming of ES cells towards the trophoblast lineage, and specifally into self-renewing TS cells, can seemingly be achieved by manipulation of transcription factors such as Cdx2 and Oct4, or modulation of signalling cascades, notably Ras signalling. Here we analyze the arising cells from such treatment in detail for the efficiency and completeness of the reprogramming process. We find that the reprogrammed cells retain an epigenetic and transcriptional memory of their ES cell origin and are not equal to bona fide trophectoderm-derived TS cells.

overall_design: RNA-seq expression analysis in conventional ES and TS cells and various ES-to-TS reprogramming models

first: Francesco

last: Cambuli

first: Alexander

last: Murray

first: Wendy

last: Dean

first: Dominika

last: Dudzinska

last: Andrews

first: Claire

last: Senner

last: Cook

first: Myriam

last: Hemberger


title: Assessment of patient-derived tumour xenografts (PDXs) as a discovery tool for cancer epigenomics [meDIP-seq]

first: Paul

last: Guilhamon

summary: Validation of methylation data for 9 osteosarcoma Patient tumours and PDXs by MeDIP followed by next generation sequencing

overall_design: 9 samples, MeDIP done with Diagenode kit and libraries prepped using NEB kit, sequenced on HiSeq 2000


title: Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast [MeDIP-Seq]

overall_design: DNA methylation analysis in conventional ES and TS cells and various ES-to-TS reprogramming models


title: RiboMeth-seq: High-throughput mapping of ribose methylations in RNA at single-nucleotide resolution

city: Copenhagen

first: Nicolai

last: Krogh

address: Blegdamsvej 3B

department: Department of Cellular and Molecular Medicine

country: Denmark

institute: University of Copenhagen

summary: The major information-carrying macromolecules in the cell, DNA, RNA, and protein, carry an additional layer of information on top of their sequence in the form of modifications of residues. The modifications provide additional functional groups and impact the structure and function of the molecules. Cellular RNA molecules contain more than 100 different modifications and are found in all domains of life and in all major classes of RNA in eukaryotic organisms. Together these modifications constitute the epitranscriptome of which two-thirds are methylations with 2’-O methylation of the ribose moiety of the nucleotide as the most abundant. Many aspects of ribose methylation are underexplored because the existing methods for their detection are laborious and can only address a few modification sites at a time.  Here, we introduce RiboMeth-Seq, a high-throughput sequencing based method and applies it to yeast ribosomal RNA. We detect all of the known as well as one new methylation site and provide evidence for hypomethylation at specific residues. Furthermore we demonstrate that many methylation events are interdependent and outline the timing of modifications during ribosome biogenesis. Our results demonstrate a novel and efficient approach to understanding of the role of modifications in ribosomal RNA folding and ribosome function. Recent evidence point to changes in ribose methylation patterns in cancer ribosomes and we anticipate that RiboMeth-Seq can be applied here as well as to other diseases in which ribosomes are affected, including the heritable ribosomopathies.

overall_design: Yeast RNA was degraded at denaturing conditions into small fragments, long enough to be mapped by sequence alignment to the reference genome (20-40 nucleotides). The fragments were ligated to RNA oligos using a tRNA ligase that was mutated to remove its kinase activity, reverse transcribed and the cDNA used as input for Ion semiconductor sequencing. The first and last nucleotides of the inserts were recorded using the full sequence for mapping and the read-ends plotted against the sequence. 2'-O-Methylated RNA positions are protected from cleavage, and read ends from such positions are therefore underrepresented compared to the surrounding positions.

first: Ulf

last: Birkedal

first: Mikkel

last: Christensen-Dalsgaard

first: Radhakrishnan

last: Sabarinathan

first: Jan

last: Gorodkin

first: Henrik

last: Nielsen


title: Chromatin occupancy and target genes of TCF7L2 in hepatocytes

city: Kuopio

first: Sami

last: Heikkinen

address: Yliopistonranta 1E

department: Institute of Biomedicine

institute: University of Eastern Finland

summary: TCF7L2 regulates multiple metabolic pathways in hepatocytes through a transcriptional network involving HNF4α

overall_design: For the identification of Tcf7l2 target genes using a RNA-seq timecourse, and for identifying the binding sites of Tcf7l2 and Hnf4a, Tcf7l2 was silenced in rat H4IIE hepatocytes using siRNA for Tcf7l2 with a scrambled siRNA as control. Treatment times for RNA-seq samples were 3, 6, 9, 12, 15, 18, 48, and 96 hours, and for ChIP-seq samples 15 h. RNA-seq timecourse was performed in duplicate or triplicate, and the ChIP-seq in duplicate for Tcf7l2 and in singlicate for Hnf4a. The H4IIE-specific transcriptome was defined from an independent set of pooled 24 h siRNA treated samples (N=3 for siRNA for Tcf7l2 and N=3 for scrambled siRNA).

first: Luke

last: Norton


title: Genes differentially expressed in Pseudomonas chlororaphis 30-84 SCV

first: DONGPING

last: WANG

summary: To better understand transcriptional changes in the SCV, RNA-seq analyses were conducted by comparing a SCV mutant to the wild-type. Transcriptomic profiling identified more than 1000 genes indicating a significant reprograming of gene expression in the SCV mutant. In accordance with observed phenotypes, genes involved in metabolism functions, motility and phenazine production were downregulated; whereas, oxidative stress and iron uptake genes were significantly upregulated.

platform_organism: Pseudomonas chlororaphis subsp. aureofaciens 30-84

overall_design: A total of 5 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas chlororaphis wild type strain (3 replicates);  Pseudomonas chlororaphis SCV mutant (2 replicates).

first: Dongping

last: Wang

first: Elizabeth

last: Pierson


title: miR-155 plays a crucial role in ALS and is an immune therapeutic target [RNA-Seq]

first: Oleg

last: Butovsky

address: 77 Avenue Louis Pasteur, Office 614

department: Center of Neurologic Diseases

institute: Brigham and Women's Hospital, Harvard Medical School

summary: Amyotrophic lateral sclerosis (ALS) is a paralytic degenerative disease of the nervous system. In the SOD1 mouse model of ALS we found loss of the molecular and functional microglia signature associated with pronounced expression of miR-155 in SOD1 mice. We also found increased expression of miR-155 in the spinal cord of ALS subjects. Genetic ablation of miR-155 increased survival in SOD1 mice and reversed the abnormal microglial and monocyte molecular signature. In addition, dysregulated proteins in the spinal cord of SOD1 mice that we identified in human ALS spinal cords and CSF were restored in SOD1G93A/miR155-/- mice. Treatment of SOD1 mice with anti-miR-155 SOD1 mice injected systemically or into the cerebrospinal fluid prolonged survival and restored the microglial unique genetic and microRNA profiles. Our findings provide a new avenue for immune based therapy of ALS by targeting miR-155.

overall_design: Total RNA was isolated from whole lumbar spinal cord homogenate from healthy control donors without known neurologic diseases and sporadic and familial ALS.


title: CpG island-mediated global gene regulatory modes in mouse embryonic stem cells

city: Austin

first: Jonghwan

last: Kim

address: 2506 Speedway NMS 4.246

department: Department of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology

institute: University of Texas at Austin

summary: In order to identify the regions occupied by transcription factors, we performed bio-ChIP followed by massive parallel sequencing (bioChIP-Seq) in mouse ES cells. A total of seven DNA transcription factors including Myc module (Myc, Max, E2F4, Tip60 and Dmap1) and Core module transcription factors (Nanog, Dax1; Kim et al., 2010) were identified. We show that the transcription factors can be classified into several classes according to their occupancies. Our data also show transcription factors in the same class co-occupy the same regulatory elements, share the common characteristics and act together.

overall_design: Identification of seven transcription factor binding sites in mES cells

first: Samuel

last: Beck


title: iCLIP seq reveals distribution of genome-wide SR protein-RNA binding

status: Public on Nov 23 2014

city: Kansas City

first: Malcolm

last: Cook

email: mec@stowers.org, malcolm.cook@gmail.com

state: MO

address: 1000 E 50th Street

department: Big Data AI

institute: Stowers Institute For Medical Research

summary: We identified the precise genome-wide binding sites for all SR proteins, using iCLIP-seq

overall_design: SR proteins were encoded on stable transgenes, transfected in S2 cells, FLAG-tag immunopurified, and the bound RNA purified and subjected to RNA-seq.  The resulting reads (CLIP tags) were aligned to the Drosophila genome and generated 38,695-5,900,000 unique CLIP tags for each SR-protein replicate.

first: Todd

last: Bradley

first: Marco

last: Blanchette


title: RNA-seq analysis of the eight Drosophila SR protein family members

summary: Using RNA-seq, we characterize the global AS regulation of the eight Drosophila SR protein family members

overall_design: RNA-seq experiments on two replicate samples from 8 individual SR protein knockdown (exptGroup=S), two replicates of simultaneous SR protein knockdown (XL6:B52 & SC35:B52) (exptGroup=D).  Each exptGroup includes duplicate of its own non-specific (NS) controls.


title: An optimized microRNA scaffold increases shRNA processing and target knockdown

first: Simon

last: Knott

state: NY

address: 1 Bungtown Rd

department: Biology

summary: Two shRNAs were placed into expression vectors harboring mir30 microRNA scaffold and an optimized scaffold where the artificial restriction sights in mir30 have been removed. After infection and selection shRNA processing was assessed by small-RNA cloning. For both shRNAs, placement into the optimized scaffold resulted in a ~two-fold increase in processing (based on smallRNA levels).

sample_organism:
Gallus gallus
Homo sapiens

platform_organism:
Gallus gallus
Homo sapiens

overall_design:
Purpose: Others have reported that the EcoRI site that was introduced to the mir30 scaffold results in decreased smallRNA processing and hence reduced target knockdown. We've developed an alternative scaffold (termed ultramir) where this site is removed. smallRNA cloning was used to determine if the movement of this sight resulted in an increase in shRNA processing.
Method: Two shRNAs (one targeting Renilla Luciferase and one targeting Human RPA3) were cloned into the original mir30 cassette the ultramir cassette. Each of the 4 constructs were infected in duplicate at single copy into cells and the cells seltected unitil infection percentages reached >90% (the shRenilla hairpin was infected into HEK293T cells and the shRPA3 construts into the Gallus gallus cell line ERC. After selection smallRNA cloning was perfromed and the amount of smallRNAs corrresponding to the two shRNAs compared to the endogenous microRNA populatlon.
Results: smallRNA levels of the two shRNAs doubled relative to the microRNA population when they were placed into the ultramir scaffold.


title: Transcriptome Comparison of Cabernet Sauvignon Grape Berries from Two Regions with Distinct Climate

status: Public on Nov 21 2014

city: Beijing

first: Run-Ze

last: Sun

address: No.20 Nanxincun, Xiangshan, Haidian District

department: Institute of Botany

institute: Chinese Academy of Sciences

summary: Primary and secondary metabolism in grape berries is under the control of complex interactions among environmental conditions, genotypes, and management practices. To obtain an interpretation from the view of transcriptome on distinct metabolite accumulation between ecologically different regions in China, next-generation sequencing technology was performed on E-L 31, 35, and 38 stages of Cabernet Sauvignon grape berries from Changli (CL, eastern) and Gaotai (GT, western). The transcript abundance of epoxycarotenoid dioxygenase and xanthoxin dehydrogenase required for ABA biosynthesis was significantly higher in the GT berries at E-L 35 and 38 stages compared with the CL berries, which may explain the relatively short maturation period of berries in the western region. Some genes required for carbohydrate metabolism, such as hexose transporter, L-idonate dehydrogenase, and phosphoenolpyruvate carboxylase, were significantly up-regulated in the CL berries in relation to the GT berries, which positively correlated with the sugar and organic acid accumulations. Pathway enrichment analysis of differentially expressed genes revealed that the CL berries had higher levels of phenylpropanoid biosynthesis at E-L 38 stage than the GT berries, which may relate to the quick fading of the GT wines because of weak co-pigmentation. This observation lays a foundation for further study concerning the molecular basis for environmental effects on berry quality formation.

overall_design: cDNA libraries generated from three developmental stages (E-L 31, 35 and 38) of Cabernet Sauvignon grape berries from Changli (CL, eastern) and Gaotai (GT, western) in China were sequenced using Illumina HiSeq™ 2000.

first: Qiu-Hong

last: Pan

first: Jun

first: Chang-Qing

last: Duan


title: SR proteins control a complex network of RNA-processing events

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

overall_design: Refer to individual Series


title: shRNA Sensor Analysis of DSIR Selected shRNAs

summary: Purpose: Generate a large, high quality database of paired shRNA efficacy/sequence datapoints.

overall_design:
Methods:Twelve shRNAs for each Refseq annotated human gene were selected based on the DSIR algorithm. Twelve batches of ~22K shRNAs (corresponding to 12 agilent chips) were then assessed for efficacy via the sensor method outlined in Fellmann et al, Mol Cell, 2011.
Conclusions: Neighboring nucleotide combinations are best at predicting shRNA efficacy.


title: Targeting of Super-Enhancer-Associated Oncogenic Transcription in MYCN-driven tumor cells by CDK7 inhibition [ChIP-Seq]

city: Boston

first: Rani

last: George

address: 450 Brookline Ave

department: Pediatric Oncology

institute: Dana Farber Cancer Institute

summary: ChIP-seq for Pol II, H3K27ac and H3K4me1 in neuroblastoma

first: Edmond

last: Chipumuro

first: Eugenio

last: Marco

first: Nicholas

last: Kwiatkowski

first: Brian

last: Abraham


title: Targeting of Super-Enhancer-Associated Oncogenic Transcription in MYCN-driven tumor cells by CDK7 inhibition


title: RNA-Seq of single cells from the mouse lumbar dorsal root ganglion

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

summary: In order to establish a consensus catalog of dorsal rott ganglion cell types, we used comprehensive transcriptome analysis of single cells for unsupervised identification and molecular classification of sensory neurons independent of any a priori knowledge of sensory subtypes.

overall_design: RNA-Seq was performed on 799 dissociated single cells dissected from the mouse lumbar dorsal root ganglion distributed over a total of nine 96-well plates

first: Dmitry

last: Usoskin

first: Peter

last: Lönnerberg

first: Patrik

last: Ernfors


title: ELAV ChIP-seq in Drosophila melanogaster embryos

city: Berkeley

first: Wei

last: Zhang

address: University of California at Berkeley

institute: UC Berkeley

summary: Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes, such as stem cell differentiation and cancer. A particularly dramatic form of APA has been documented in the developing nervous system of flies and mammals, whereby a variety of neurogenic genes undergo coordinate extension of their 3’ UTRs. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation (poly(A)) sites, thereby fostering the formation of 3’ extensions that can reach 12 kb in length. Here, we present evidence that paused Pol II plays an important role in the selective recruitment of ELAV to elongated genes. Replacing native promoters of elongated genes with heterologous promoters blocks normal 3’ extension in the nervous system, while native promoters can induce 3’ extension in ectopic tissues expressing ELAV. Computational analyses suggest that the promoter regions of elongated genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ELAV ChIP-Seq assays indicate pervasive binding to the promoter regions of extended genes. Our study provides the first evidence for a regulatory link between promoter-proximal pausing and APA.

overall_design: ELAV ChIP-Seq assays were conducted with nuclei obtained from 6-8 hr and 10-12 hr embryos

first: Katarzyna

last: Oktaba

first: Thea

middle: S

last: Lotz

first: David

last: Jun

first: Sandra

last: Lemke

first: Samuel

last: Ng

first: Emilia

last: Esposito

first: Michael

last: Levine

first: Valérie

last: Hilgers


title: Dysregulation of microRNAs in neurodegeneration

city: New York

first: Ismael

last: Santa Maria

address: 630W 168TH STREET

department: Pathology & Cell Biology

institute: Columbia University

summary: This dataset contains small RNA-seq data from six Alzheimer disease, three tangle-predominant dementia and seven control brains.

overall_design: Total RNA was isolated from human post-mortem autopsy brain and small RNA-seq was performed as described [Hafner, M., Renwick, N., Brown, M., Mihailovic, A., Holoch, D., Lin, C., Pena, J.T., Nusbaum, J.D., Morozov, P., Ludwig, J., et al. RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA. 2011; 17(9):1697-1712]

last: Santa-Maria

first: Neil

last: Renwick

first: Thomas

last: Tuschl

first: John

last: Crary


title: Targeting miR-155 restores abnormal microglia and attenuates disease in SOD1 mice

first: Oleg

last: Butovsky


title: Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci

city: Lausanne

first: Nicolas

last: Lonfat

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

department: School of Life Sciences

institute: EPFL - Ecole Polytechnique Fédérale de Lausanne

type: Genome binding/occupancy profiling by genome tiling array


title: Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci (RNA-seq)

summary: Hox genes are required during the morphogenesis of both vertebrate digits and external genitals.We investigated whether transcription in such distinct contexts involves a shared enhancer-containing landscape. We show that the same regulatory topology is used, yet with some tissue-specific enhancer-promoter interactions, suggesting the hijacking of a regulatory backbone from one context to the other. In addition, comparable organizations are observed at both HoxA and HoxD clusters, which separated through genome duplication in an ancestral invertebrate animal.We propose that this convergent regulatory evolution was triggered by the pre-existence of some chromatin architecture, thus facilitating the subsequent recruitment of the appropriate transcription factors. Such regulatory topologies may have both favored and constrained the evolution of pleiotropic developmental loci in vertebrates.

overall_design: RNA-seq from mouse E12.5 digits cells and mouse E15.5 genital tubercle cells.

first: N

first: T

last: Montavon

first: F

last: Darbellay

first: S

last: Gitto

first: D

last: Duboule


title: NOTCH1 activation in breast cancer confers sensitivity to inhibition of SUMOylation

city: Vienna

first: Stefan

last: Kubicek

address: Lazarettgasse 14

institute: CeMM

summary: Breast cancer is genetically heterogeneous, and recent studies have underlined a prominent contribution of epigenetics to the   development of this disease. To uncover new synthetic lethalities with known breast cancer oncogenes, we screened an  epigenome-focused short hairpin RNA library on a panel of engineered breast epithelial cell lines. Here we report a selective  interaction between the NOTCH1 signaling pathway and the SUMOylation cascade. Knockdown of the E2-conjugating enzyme  UBC9 (UBE2I) as well as inhibition of the E1-activating complex SAE1/UBA2 using ginkgolic acid impairs the growth of NOTCH1-activated breast epithelial cells. We show that upon inhibition of SUMOylation NOTCH1-activated cells proceed slower through the  cell cycle and ultimately enter apoptosis. Mechanistically, activation of NOTCH1 signaling depletes the pool of unconjugated small  ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 leading to increased sensitivity to perturbation of the SUMOylation cascade.  Depletion of unconjugated SUMO correlates with sensitivity to inhibition of SUMOylation also in patient-derived breast cancer cell  lines with constitutive NOTCH pathway activation. Our investigation suggests that SUMOylation cascade inhibitors should be  further explored as targeted treatment for NOTCH-driven breast cancer.

overall_design: We treated MCF10A and NOTCH1 cells with either DMSO or ginkgolic acid 30 uM for 3 days. Two replicates have been analysed for each condition.

first: Marco

last: Licciardello

first: Markus

middle: K

last: Müllner

first: Gerhard

last: Dürnberger

first: Claudia

last: Kerzendorfer

first: Bernd

last: Boidol

last: Trefzer

first: Sara

last: Sdelci

first: Tiina

last: Berg

last: Penz

last: Schuster

first: Christoph

last: Bock

first: Robert

last: Kralovics

first: Giulio

last: Superti-Furga

first: Jacques

last: Colinge

first: Sebastian

middle: M

last: Nijman


title: MethylC-Seq of Ws background wild-type (WT), ape1l-1 (Ws background), arp-1 (Col-0 background) and zdp-1 (Col-0 background) mutants

city: West Lafayette

first: Jian-Kang

last: Zhu

state: IN

address: 625 Agriculture Mall Dr.

department: Department of Horticulture and Landscape Architecture

institute: Purdue University

summary: Using MethylC-Seq to provide single-base resolution of DNA methylation status in Ws background wild-type (WT), ape1l-1 (Ws background), arp-1 (Col-0 background) and zdp-1 (Col-0 background) mutants

overall_design: MethylC-Seq: 1 WT and 3 mutants examined, Ws background wild-type (WT), ape1l-1 (Ws background), arp-1 (Col-0 background) and zdp-1 (Col-0 background) mutants

first: Yan

last: Li

first: Weiqiang

last: Qian

first: Kai

last: Tang


title: Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

first: Krassimira

middle: I

last: Botcheva

address: One Cyclotron Rd MS977

department: Cell and Molecular Biology

institute: Lawrence Berkeley National Laboratory

summary: We mapped the genomic binding sites of the tumor suppressor protein p53 in the human colorectal cancer cell line HCT116 and report here that the binding patterns of endogenous wild type p53 differed significantly between the genomes of the cancer cell line HCT116 and the normal human IMR90 fibroblasts (GSE31558) under the same experimental conditions (6 hr treatment with 5-fluorouracil). p53 binding differences affect promoter regions, CpG islands and major families of human repeat elements such as LTR, LINE and SINE. While p53 genomic binding sites residing in repeats have been reported before, we show here that the fraction of the p53 genomic binding sites residing in different repeat families differs between the normal and cancer human cell lines. We confirm that the p53 genomic binding sites in HCT116 cells are excluded from CpG islands, an observation we made previously based on analysis of data reported by others. While the p53 ability to elicit stress-specific and cell-type-specific responses is well documented, how this specificity is established, at the level of binding to the genome and/or during post-binding events, represents an open question. Our data indicate that p53 binding to the human genome is cell line-specific and highly selective. The differences in the p53 genome-wide binding patterns between the cancer cell line HCT116 and the normal cell line IMR90, namely exclusion from CpG islands and enrichment at repeats in HCT116, likely reflect cancer-associated epigenetic changes in the chromatin.

overall_design: Identification of genomic p53 binding sites in HCT116 cells by ChIP-seq.

first: Sean

last: McCorkle


title: Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci (4C-seq)

first: Nicolas

last: Lonfat

overall_design: Circular Chromosome Conformation Capture (4C-seq) samples from mouse digit cells and mouse genital tubercle cells performed as previously described (Noordermeer et al. 2011)

first: N

first: T

last: Montavon

first: F

last: Darbellay

first: S

last: Gitto

first: D

last: Duboule


title: Circadian Enhancers Coordinate Multiple Phases of Rhythmic Gene Transcription In Vivo

city: Philadelphia

first: Bin

last: Fang

address: 3400 Civic Center Blvd, Bldg 42

institute: University of Pennsylvania

summary: Mammalian transcriptomes display complex circadian rhythms with multiple phases of gene expression that cannot be accounted for by current models of the molecular clock.  We have determined the underlying mechanisms by measuring nascent RNA transcription around the clock in mouse liver.  Unbiased examination of eRNAs that cluster in specific circadian phases identified functional enhancers driven by distinct transcription factors (TFs).  We further identify on a global scale the components of the TF cistromes that function to orchestrate circadian gene expression.  Integrated genomic analyses also revealed novel mechanisms by which a single circadian factor controls opposing transcriptional phases.  These findings shed new light on the diversity and specificity of TF function in the generation of multiple phases of circadian gene transcription in a mammalian organ.

overall_design: Nascent RNA transcripts in mouse liver were profiled at 8 time points of the 24 hour light-dark cycle. Static state mRNAs in WT and Rev-erbA -/- mice were profiled using microarray (GSE59460).

first: Logan

last: Everett

first: Jennifer

last: Jager

first: Erika

last: Briggs

last: Armour

first: Dan

last: Feng

first: Ankur

last: Roy

first: Zachary

last: Gerhart-Hines

first: Zheng

last: Sun

first: Mitchell

middle: A

last: Lazar


title: RNA sequencing of bone marrow CD34+ cells from myelodysplastic syndrome patients with and without SF3B1 mutation and from healthy controls

status: Public on Nov 22 2014

city: Oxford

first: Hamid

last: Dolatshad

address: John Radcliffe Hospital

department: RDM-NDCLS

institute: University of Oxford

summary: The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34+ cells from MDS patients with SF3B1 mutations using RNA-sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared to wildtype cases include genes involved in MDS pathogenesis (ASXL1, CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7, SLC25A37) and RNA splicing/processing (PRPF8, HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. Our data indicate that SF3B1 plays a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.

overall_design: RNA-Seq was performed to compare the transcriptome of bone marrow CD34+ cells from eight MDS patients with SF3B1 mutation, four MDS patients with no known splicing mutation and five healthy controls.

first: Andrea

last: Pellagatti


title: Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid.

city: Norwich

first: Ghanasyam

last: Rallapalli

address: Norwich Research Park

institute: The Sainsbury Laboratory

summary: Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

overall_design:
Three weeks old Arabidopsis (Col-0) plants were inoculated with either the avirulent isolate Emoy2 (incompatible interaction) or the virulent isolate Waco9 (compatible interaction) of Hpa, and infected plants were harvested at 1, 3 and 5 days post-inoculation (dpi) for total RNA extraction. mRNA profiles were generated by deep sequencing on Illumina GAIIx using EXPRSS tag-seq protocol. Each biological replicate set of samples were sequenced as one batch (biorep1, biorep2 and biorep3 in filenames) and each batch was sequenced in 4 individual Illumina flowcell lanes (lane1 to lane4 in filenames). Four sequecing lanes of each three biological repliates resulted in 12 sequencing libraries. Emoy2 1, 3 and 5 dpi samples and Waco9 1 dpi samples were made two different codes (lib1 and lib2 in filenames) to generate higher depth of sequencing data.

Each biological replicate set of samples were sequenced as one batch and each batch was sequenced in 4 individual Illumina flowcell lanes. Four sequecing lanes of each three biological repliates resulted in 12 sequencing libraries. 'The unassigned read_*' samples are for 'nobarcode_biorep[x]_lanne[x].fq'  file containing sequences unassigned to any barcode from respective bioreplicate sequencing lanes.

first: Shuta

last: Asai

first: Sophie

middle: J

last: Piquerez

first: Marie-Cécile

last: Caillaud

first: Oliver

last: Furzer

first: Naveed

last: Ishaque

first: Lennart

last: Wirthmueller

first: Georgina

last: Fabro

first: Ken

last: Shirasu

first: Jonathan

middle: D

last: Jones


title: The Nuclear Immune Receptor RPS4 Is Required for RRS1SLH1-Dependent Constitutive Defense Activation in Arabidopsis thaliana.

summary: Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific “avirulent” pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.

overall_design:
Arabidopsis No-0 and slh1 plants were grown for 4 weeks at 28°C after germination on MS plate. Plants were transferred to 19°C growth chamber at the beginning of the light cycle and samples were harvested at 0 h, 9 h, 12 h, 16 h and 24 h after transfer for total RNA extraction. mRNA profiles were generated by deep sequencing on Illumina GAIIx using EXPRSS tag-seq protocol.
Please note that all 16 samples submitted for this study were sequenced in one lane of Illumina GAIIx  and the "No-0_slh1_tempshift_nobarcode.fq" (linked to 'unassigned reads' sample) contains unassigned sequence reads, once sample de-multiplexing has been carried out based on barcode.

first: Kee

last: Sohn

first: Cécile

last: Segonzac

first: Panagiotis

middle: F

last: Sarris

first: Joo

middle: Y

last: Woo

first: Simon

last: Williams

first: Toby

last: Newman

first: Kyung

last: Paek

first: Bostjan

last: Kobe