title: A variety of Dicer substrates in human and C. elegans (HEK RNA-seq)

status: Public on Nov 20 2014

city: Berlin

first: Marvin

last: Jens

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

department: Systems Biology of Gene Regulatory Elements

country: Germany

institute: Max Delbrueck Center for Molecular Medicine

summary: The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/microRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer binding sites. We find known and hundreds of novel miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets. Interestingly, most Dicer binding sites reside on mRNAs/lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside the miRNA pathway.

overall_design: Regulatory impact of Dicer binding was assessed by knock down experiments in human HEK293 cells. Drosha knockdown and mock transfections were used as controls. Knockdown was performed with two independent siRNAs each. In total 5 samples.

first: Agnieszka

last: Rybak-Wolf

first: Yasuhiro

last: Murakawa

first: Markus

last: Landthaler

first: Nikolaus

last: Rajewsky


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

status: Public on Nov 21 2014

city: Norwich

first: Ghanasyam

last: Rallapalli

address: Norwich Research Park

country: United Kingdom

institute: The Sainsbury Laboratory

type: Expression profiling by high throughput sequencing

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: Five weeks old Arabidopsis (Ws-2 and rrs1-1) leaves were infiltrated with Pf0-1 carrying PopP2 or PopP2C321A in pEDV6 and samples were collected at 2, 4, 6 and 8 hours post infiltration (hpi); mRNA profiles were generated in triplet, by deep sequencing on Illumina GAIIx using EXPRSS tag-seq protocol.

first: Jonathan

middle: D

last: Jones


title: In vivo activation of a conserved microRNA program induces robust mammalian heart regeneration (RNA-Seq)

city: La Jolla

first: Christopher

last: Benner

state: California

address: 9500 Gilman Dr. MC 0640

department: Medicine

country: USA

institute: University of California, San Diego (UCSD)

summary: Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here, we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c, and their protein targets smarca5 and fntb, as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response following myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof-of-concept for identifying and activating conserved molecular programs to regenerate the damaged heart.

overall_design: RNA-Seq expression profiles of rat cardiomyocytes after knockdown of miR-99/100 and Let-7 miRNAs

first: Aitor

last: Aguirre

first: Juan

middle: C

last: Belmonte


title: Transcriptomic profiling of apple seeds

city: Milan

first: Sergio

last: Ferrero

address: Via Celoria 26

department: Dipartimento di Bioscienze

country: Italy

institute: Università degli Studi di Milano

summary: Comparison of seed of two different types of developing fruits in apple: central and lateral. Our objective is to find transcriptomic signatures that allow to explain the physiological drop of young lateral fruitlets

overall_design: Apple seeds transcriptomes were generated by deep sequencing by triplicate from seeds collected from central and lateral fruitlets at 20 days after petal fall (DAPF)

first: Ferrero

last: Sergio

first: Masiero

last: Simona

first: Colombo

last: Lucia


title: Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (CEL small RNA)

type: Non-coding RNA profiling by high throughput sequencing

summary: Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and human. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis.

overall_design: small RNAs were extracted from total RNA of lysed worms. Standard small RNA sequencing was performed to sequence 5' monophosphate bearing small RNAs. Alternatively, 5'polyphosphatase from Epicenter was used to catch also tri-phosphate bearing secondary endo-siRNAs.


title: Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (CEL RNA-Seq)

summary: Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and C.elegans. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis.

overall_design: Regulatory impact of Dicer binding was assessed by knock down experiments in human HEK293 cells and C.elegans. Drosha knockdown and mock transfections were used as controls. In total 6 samples (3 human, 3 nematode)


title: Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (small RNA AGO-IP)

overall_design: Argonaute loaded small RNAs were extracted from FLAG:AGO2 and FLAG:AGO3 expressing HEK293 cells. Small RNA was purified and length selected (see supplementary methods).


title: Transcriptome analysis in mouse late spermatocyte deficient in pachytene piRNAs (MIWI-immunoprecipitated RNAs)

city: NewHaven

first: Toshiaki

last: Watanabe

state: CT

address: 10 Amistad

institute: Yale University

summary: The eukaryotic genome has vast intergenic regions containing transposons, pseudogenes and other repetitive sequences. They produce numerous long non-coding RNAs (lncRNAs) and PIWI-interacting RNAs (piRNAs), yet the functions of the vast intergenic regions remain largely unknown. Mammalian piRNAs are abundantly expressed in late spermatocytes and round spermatids. Their expression coincides with the widespread expression of lncRNAs from the genome of these cells. Here, we show that piRNAs mediate the degradation of a large number of mRNAs and lncRNAs in mouse late spermatocytes. In particular, they have a large impact on the lncRNA transcriptome, as a quarter of lncRNAs expressed in late spermatocytes are upregulated in mice deficient in piRNA pathway. Furthermore, our genomic and in vivo functional analyses reveal that retrotransposon sequences in the 3´UTR of mRNAs are potentially targeted by piRNAs for degradation. Similarly, a large number of spermatogenic cell-specific lncRNAs are degraded by piRNAs via retrotransposon sequences. Moreover, we show that pseudogenes regulate mRNA stability via the piRNA pathway. The degradation of mRNAs and lncRNAs by piRNAs requires MIWI and, at least in part, depends on its slicer activity. Together, these findings reveal the presence of the highly complex and global RNA regulatory network mediated by piRNAs with retrotransposons and pseudogenes as regulatory sequences.

overall_design: RNA-seq of MIWI-immunoprecipitated RNAs from Late spermatocytes in Miw+/-, MiwiADH/-, Miwi-/-

first: T

first: M

last: Zhong

first: E

last: Cheng

first: H

last: Lin


title: A variety of Dicer substrates in human and C. elegans (HEK PAR-CLIP)

first: Marvin

last: Jens

overall_design: PAR-CLIP basically as described previously (Hafner et al. 2010).

first: Agnieszka

last: Rybak-Wolf

first: Yasuhiro


title: Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (HEK RNA-Seq)

overall_design: Regulatory impact of Dicer binding was assessed by knock down experiments in human HEK293 cells. Drosha knockdown and mock transfections were used as controls. In total 3 samples.

last: Murakawa

first: Markus

last: Landthaler

first: Nikolaus


title: Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (CEL PAR-CLIP)

overall_design: In vivo PAR-CLIP basically as described previously (Jungkamp et al. 2011). FLAG tagged DCR-1 expressing (rescue strain) and 4SU labeled animals were UV irradiated at young adult stage. Worm lysate (NP40) was treated with RNase T1 and RNase V1. After that, PAR-CLIP proceeded as described in Hafner et al. 2010.

last: Rajewsky


title: Single-cell transcriptome analysis of secretagogin mRNA expressing cells from the mouse hypothalamic paraventricular nucleus

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

summary: The molecular mechanism regulating phasic corticotropin-releasing hormone (CRH) release from parvocellular neurons (PVN) remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin’s Ca2+ sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone.

overall_design: single cells from the PVN region juvenile (21-28 days) mice were dissected and subject to whole transcriptome analysis

first: Roman

last: Romonov


title: Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution

city: Stanford

first: ENCODE

last: DCC

state: CA

address: 300 Pasteur Dr

institute: ENCODE DCC

type: Genome binding/occupancy profiling by high throughput sequencing

summary: To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I–hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

first: Jeffrey

last: Vierstra

first: Eric

last: Rynes

first: Richard

last: Sandstrom

first: Robert

middle: E

last: Thurman

first: Miaohua

last: Zhang

first: Theresa

last: Canfield

first: Peter

middle: J

last: Sabo

first: Rachel

last: Byron

first: R

middle: S

last: Hansen

first: Audra

middle: K

last: Johnson

first: Shinny

last: Vong

first: Kristen

last: Lee

first: Daniel

last: Bates

first: Fidencio

last: Neri

first: Morgan

last: Diegel

first: Erika

last: Giste

last: Haugen

first: Douglas

last: Dunn

last: Humbert

first: Matthew

last: Wilken

first: Steven

last: Josefowicz

last: Samstein

first: Kai-Hsin

last: Chang

first: Dana

last: Levassuer

first: Christine

last: Disteche

first: Marella

last: De Bruijn

first: Thomas

middle: A

last: Rey

first: Arthur

last: Skoultchi

first: Aleksander

last: Rudensky

first: Stuart

middle: H

last: Orkin

first: Thalia

last: Papayannopoulou

first: Piper

last: Treuting

first: Licia

last: Selleri

first: Rajinder

last: Kaul

last: Bender

first: Mark

last: Groudine

first: John

last: Stamatoyannopoulos


title: A variety of Dicer substrates in human and C. elegans (CEL RNA-seq)

first: Marvin

last: Jens

overall_design: Regulatory impact of Dicer binding was assessed by knock down experiments in human HEK293 cells and Caenorhabditis elegans. Drosha knockdown and mock transfections were used as controls. In total 6 samples (3 human, 3 nematode)

first: Agnieszka

last: Rybak-Wolf

first: Yasuhiro

last: Murakawa

first: Markus

last: Landthaler

first: Nikolaus

last: Rajewsky


title: Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (HEK PAR-CLIP)


title: Genome-Wide Landscapes of DNA Methylomes in Bovine Muscle Tissue

city: Yangling

first: Yongzhen

last: Huang

state: Shaanxi

address: No.22 Xinong Road

department: College of Animal Science and Technology

country: China

institute: Northwest A&F University

type: Methylation profiling by high throughput sequencing

summary: DNA methylation is a key epigenetic modification in mammals, have essential and important roles in muscle development. Here, using cattle as a model, we investigate the systematic association between DNA methylation and muscle development. We sample longissimus thoracis tissues from a famous elite native breed of Chinese Qinchuan cattle living within comparable environments under fetal and adult stages. Using methylated DNA immunoprecipitation sequencing (MeDIP-Seq) , we generate and provide a genome-wide landscapes of DNA methylomes for muscle studies. The analysis shows global similarity and difference among fetal and adult stage and identifies the differentially methylated regions. The differentially methylated regions in promoters are highly associated with muscle development via expression repression of both known muscle-related genes and novel genes. This comprehensive map provides a solid basis for exploring epigenetic mechanisms of muscle growth and development.

overall_design: Examination of 2 different developmental stages of the Longissimus Muscle

first: Yong-Zhen

first: Hong

last: Chen


title: Transcriptome analysis in mouse late spermatocyte deficient in pachytene piRNAs (Mov10l1 fl/+ and Mov10l1 s fl/-)

first: Toshiaki

last: Watanabe

summary: In animal germline cells, Piwi-interacting RNAs (piRNAs) silence retrotransposons through post-transcriptional and transcriptional mechanisms. However, little is known, especially in mammals, about the functions of piRNAs beyond retrotransposon suppression1-5. In mammalian spermatocytes, piRNAs are known to be abundantly expressed6-10. Here, we show that a subset of coding and noncoding RNAs in mouse spermatocytes is degraded by the piRNA pathway. By analyzing the germline trasnscriptome of mice deficient in piRNA biogenesis, we identify hundreds of mRNAs as direct targets of piRNAs. Remarkably, the 3' untranslated region (UTR) of the mRNAs up-regulated in the piRNA pathway mutants are highly enriched with retrotransposon sequenes, implying that these sequences serve as regulatory elements for piRNA-mediated regulation. Furthermore, deficiencies of piRNAs derived from pseudogenes result in increased mRNA levels of their cognate genes, indicating that pseudogenes regulate their functional cognates via piRNAs. Moreover, we identify a large population of testis-enriched long intergenic noncoding RNAs (lincRNAs), some of which are also degraded by the piRNA pathway. Collectively, our results reveal that the piRNA pathway regulates the expression of both mRNAs and lincRNAs in addition to retrotransposon RNAs during meiosis and the key role of retrotransposons and pseudogenes, two major types of genomic sequences, in this regulation by acting as piRNA sources and/or regulatory elements in target RNAs.

overall_design: Late spermatocyte mRNA profiles of Mov10l1 fl/+ and Mov10l1 s fl/- were analyzed by deep sequencing, in triplicate, using Illumina HiSeq.

first: T

first: M

last: Zhong

first: E

last: Cheng

first: H

last: Lin


title: A variety of Dicer substrates in human and C. elegans (HEK small RNA)

overall_design: small RNAs were extracted from total RNA. Standard small RNA sequencing was performed to sequence 5' monophosphate bearing small RNAs. This was performed for siRNA knockdowns of DICER1 or DROSHA, with two independent siRNAs each, and for control cells. (5 samples in total)


title: Spermatogenesis

overall_design: Refer to individual Series


title: Transcriptome analysis during mouse testis development

overall_design: Transcriptome of  7 developmental stage during mouse testis development were analyzed by deep sequencing using Illumina HiSeq.

last: Watanable


title: Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates

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

sample_organism:
Caenorhabditis elegans
Homo sapiens

platform_organism:
Caenorhabditis elegans
Homo sapiens


title: piRNA analysis in Stambp-ps1 mutant mouse testes

overall_design: Small RNAs in Stambp-ps1 mutant testes were sequenced using Illumina HiSeq.


title: Genome-wide analysis of leafbladeless1-regulated and phased small RNAs provides new insights into the tas3 pathway in maize-

city: Cold Spring Harbor

first: Molly

last: Gale Hammell

state: NY

address: 1 Bungtown Rd

department: Quantitative Biology

institute: Cold Spring Harbor Lab

summary: Maize LEAFBLADELESS1 (LBL1) and Arabidopsis SUPPRESSOR OF GENE SILENCING3 (SGS3) play orthologous roles in the biogenesis of 21 nucleotide trans-acting short-interfering RNAs (tasiRNAs). The phenotypes conditioned by mutation of lbl1 and SGS3 are, however, strikingly different, suggesting that the activities of these small RNA biogenesis components, or the tasiRNAs and their targets might not be entirely conserved. To investigate the basis for this phenotypic variation, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects all major classes of small RNAs, and reveal unexpected crosstalk between tasiRNA biogenesis and other small RNA pathways regulating miRNAs, retrotransposons, and DNA transposons. We further identified genomic regions generating phased siRNAs, including numerous loci generating 22-nt phased small RNAs from long hairpin RNAs or overlapping antisense transcripts not previously described in other plant species. By combining both analyses, we identified nine TAS loci, all belonging to the conserved TAS3 family. Contrary to other plant species, no TAS loci targeted by a single miRNA were identified. Information from target prediction, RNAseq, and PARE analyses identified the tasiARFs as the major functional tasiRNAs in the maize vegetative apex where they regulate expression of ARF3 homologs. As such, divergence in TAS pathways is unlikely to account for the distinct phenotypes of tasiRNA biogenesis mutants in Arabidopsis and maize. Instead, the data suggests variation in the spatiotemporal regulation of ARF3, or divergence in its function, as a plausible basis for the dramatic phenotypic differences observed upon mutation of SGS3/lbl1 in Arabidopsis and maize.

overall_design:
An analysis of tasiRNA biogenesis, activity, and contribution to developmental phenotypes in the maize leaf. Data generated includes small RNA sequencing data and mRNA sequencing data.
All data was generated in both wild type and lbl1 mutant maize leaf apices. Three replicates were generated for each genotype for the small RNA data. Two of these replicates were also used for the RNA-seq data.

first: Marcela

last: Dotto

first: Milo

last: Aukerman

first: Mary

last: Beatty

last: Hammell

first: Marja

last: Timmermans


title: Epigenomic profiling of Acute myeloid leukemia subtypes from primary tumor samples

city: New York

first: Ari

last: Melnick

state: New York

address: 1305 York Ave.

institute: Weill Cornell Medical College

summary: Here we used Illumina NGS for high-throughput profiling of the DNA methylome(ERRBS) and hydroxymethylome(hMe-Seal) of primary tumor samples with Acute Myeloid Leukemia(AML). The data can be used to compare hydroxymethylation and methylation patterns from different AML subtypes and normal bone marrow samples.

overall_design: We have sequenced 4 subtypes of AML with hydroxymethylation decrease and 1 subtype with no decrease. We have sequenced 2-5 primary tumor samples for each subtype, and comprated the epigenomic profiles ( ERRBS and hMe-Seal ) of hydroxymethylation deficient subtypes to the control subtype and normal bone marrow samples.

first: Raajit

last: Rampal

first: Altuna

last: Akalin

first: Jozef

last: Madzo

first: Christopher

last: Mason

first: Lucy

last: Godley

middle: M

first: Ross

middle: L

last: Levine

first: Maria

last: Figueroa


title: Transcriptome analysis during mouse spermatogenic cell development

first: Toshiaki

last: Watanabe

overall_design: mRNAs in leptotene/zygotene spermatocytes, early-pachytene spermatocytes, mid-pachytene spermatocytes, late pachytene/diplotene spermatocytes and round spermatids were analyzed by deep sequencing using Illumina HiSeq.

first: T

last: Zhong

last: Cheng


title: RNA-Seq analysis comparing p53-null versus ΔNp63Δ/Δ;p53-null or ΔNp73Δ/Δ;p53-null thymic lymphoma tumors

status: Public on Nov 19 2014

city: Tampa

first: Elsa

middle: Renee

last: Flores

state: FL

address: 12902 USF Magnolia Drive

department: Department of Molecular Oncology

institute: Moffit Cancer Center

summary: We performed an RNA-Seq analysis comparing thymic lymphoma tissues from the p53-null(n=2) and ΔNp63Δ/Δ;p53-/- (n=3) or ΔNp73Δ/Δ;p53-/-(n=3). Mice at 10 weeks of age were injected with either Ad-mCherry or Ad-CRE-mCherry to delete ΔNp63/ΔNp73 in the thymic lmyphomas. We aimed to test by deleting the DNp63/DNp73 in these p53-deficient tumors will mediate tumor regression and analyze the expression profile of the genes

overall_design: Examination of thymic lymphoma tissues in 3 different genotypes (p53-/- vs ΔNp63Δ/Δ;p53-/- or ΔNp73Δ/Δ;p53-/-)

first: Avinashnarayan

last: Venkatanarayan

middle: R


title: Transcriptome analysis in mouse early round spermatids deficient in pachytene piRNAs (Miwi+/- and -/-)

overall_design: Early round spermatid mRNA profiles of Miwi+/- and -/- were analyzed by deep sequencing, in triplicate, using Illumina HiSeq.

last: Watanable

first: M

first: E

first: H

last: Lin


title: TDP-1, the C. elegans ortholog of TDP-43, limits the accumulation of double stranded RNA

city: Boulder

last: Link

state: Colorado

address: 1725 Pleasant st

department: Integrative Physiology

institute: University of Colorado Boulder

summary: C. elegans mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA binding protein TDP-43, display only mild phenotypes. Nevertheless, transcriptome sequencing revealed that many RNAs were altered in accumulation and/or processing in the mutant. Analysis of these transcriptional abnormalities demonstrates that a primary function of TDP-1 is to limit formation or stability of double-stranded RNA. Specifically, we found that deletion of tdp-1: 1) preferentially alters the accumulation of RNAs with inherent double stranded structure (dsRNA); 2) increases the accumulation of nuclear dsRNA foci, 3) enhances the frequency of adenosine-to-inosine RNA editing, and 4) dramatically increases the amount of transcripts immunoprecipitable with a dsRNA-specific antibody, including intronic sequences, RNAs with antisense overlap to another transcript, and transposons. We also show that TDP-43 knockdown in human cells results in accumulation of dsRNA , indicating that suppression of dsRNA is a conserved function of TDP-43 in mammals. Altered accumulation of structured RNA may account for some of the previously described molecular phenotypes (e.g., altered splicing) resulting from reduction of TDP-43 function.

overall_design: 24 samples: 3 tdp-1 polyA samples with 3 N2 controls, 3 tdp1J2 immunoprecipitated samples and tdp1 total RNA input with 3 N2 J2 immunoprecipitated controls (with N2 input), 3 tdp1 total RNA samples for RNA editing analysis with 3 N2 total RNA controls and an adr-2 mutant control, 2 tdp1 CHIPseq samples with RNAsecontrol.

first: Tassa

last: Saldi

first: Peter

last: Ash

first: Gavin

last: Wilson

first: Patrick

last: Gonzales

first: Alfonso

last: Garrido-Lecca

first: Christine

last: Roberts

first: Vishantie

last: Dostal

first: Tania

middle: F

last: Gendron

first: Lincoln

last: Stein

first: Thomas

last: Blumenthal

first: Leonard

last: Petrucelli


title: Identification of the cellular RNAs bound by MOV10

city: Urbana

first: Stephanie

last: Ceman

state: IL

address: 601 S. Goodwin Ave

department: Cell and Developmental Biology

institute: University of Illinois-Urbana Champaign

summary: Using the iCLIP protocol we have identified the cellular RNA entities that are bound by MOV10. We report the location and sequence of the MOV10 binding region on each RNA entity.

overall_design: To identify the RNAs that bound MOV10, we UV-cross-linked HEK293F cells and immunoprecipitated with an irrelevant antibody (ir or "control") followed by a MOV10-specific antibody (MOV10) to isolate associated RNAs after stringent washing.

first: Miri

last: Kim

first: Phillip

last: Kenny

first: Radhika

last: Khetani

first: Mary

last: Arcila

first: Kenneth

last: Kosik


title: Transcriptional response to zebularine and mitomycin C (MMC) in wild type and ATR deficient Arabidopsis thaliana plants

status: Public on Nov 18 2014

city: Olomouc - Holice

first: Ales

last: Pecinka

address: Slechtitelu 31

department: Centre of Plant Structural and Functional Genomics

country: Czech Republic

institute: Institute of Experimental Botany

summary: Zebularine is a non-methylable cytidine analog that is used in epigenetic and cancer research. Its application has been shown to activate several transcriptionally silenced genetic elements; however, extent of this activation and other effects on the transcriptome are unknown. Here, we show that zebularine treatment induces preferentially genes associated with DNA damage repair response and this activation is strongly ATR-dependent. The set of up- and down-regulated genes after 24 h zebularine treatment is almost fully contained in the set of genes with changed transcription in response to 24 h MMC treatment. We identified only few genes that were up-regulated in common by longer (5 days) zebularine treatment and loss of epigenetic control in ddm1 mutant. Our results suggest that zebularine does not induce global activation of targets of transcriptional gene silencing and indicate previously unanticipated DNA damaging effects associated with zebularine-treatment.

overall_design: Examination of transcriptional changes in response to mock, 24 h and 5 days 20 µM zebularine and 24 h 10 µM MMC treatment in Arabidopsis wild-type and/or atr-2.

first: Chun

last: Liu

first: Andreas

last: Finke


title: FMRP-associated MOV10 facilitates and antagonizes miRNA-mediated regulation

summary: The fragile X mental retardation protein FMRP is an RNA binding protein that regulates translation of its bound mRNAs through incompletely defined mechanisms. FMRP has been linked to the microRNA pathway and we show here that it is associated with MOV10, a putative helicase that is also associated with the microRNA pathway.  We show that FMRP associates with MOV10 in an RNA-dependent manner and facilitates MOV10-association with RNAs in brain. We identified the RNA sequences recognized by MOV10 using iCLIP and found an increased number of G-quadruplexes in the CLIP sites. We provide evidence that MOV10 facilitates microRNA-mediated translation regulation and also has the novel role of increasing the expression of a subset of RNAs by sterically hindering Argonaute2 association. In summary, we have identified a new mechanism for FMRP-mediated translational regulation through its association with MOV10.

overall_design: Comparison of MOV10 siRNA knockdown, irrelevant siRNA control and MOV10 overexpression on total RNA levels


title: Dissecting non-coding and pathogen RNA-protein interactomes

first: Ryan

middle: Alexander

last: Flynn

address: 380 Roth Way, Room 265

institute: Stanford University

summary: RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and non-human RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and non-human transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2 bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

overall_design: Characterization of non-coding and pathogen RNA-protein interactions using an automated computational pipeline and improved iCLIP biochemistry

first: Lance

last: Martin

first: Howard

middle: Y

last: Chang


title: The permanent reduction of TIA proteins molds expression transcriptome in HeLa cells

city: Cantoblanco

first: Juan

middle: Carlos

last: Oliveros

state: Madrid

address: Darwin 3

country: Spain

institute: CNB, CSIC

summary: This study provides, for the first time, TIA1 and TIAR linked-transcriptomic analysis by using RNA-Seq next-generation sequencing technology. Illumina RNA-Seq was used to survey transcriptome profiles from permanent TIA1- and TIAR-(shRNA-mediated) deficient HeLa cells. Analysis of the transcriptomes with the Cufflinks tool revealed that differentially expressed genes, isoforms produced by alternative splicing and/or promoter usage as well as microRNAs generated a great transcriptomic heterogeneity which might reflect the complexity linked to these cell phenotypes. The data of differential expression were validated by using genome-wide microarrays and QPCR analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes term enrichment analysis revealed over-representation of genes associated with cell differentiation, multicellular organismal development, signal transduction, axon guidance and cell adhesion and under-representation of genes associated with positive regulation of migration, cell adhesion, response to organic substance, prostaglandin metabolic process and blood coagulation. Taken together, these results indicate that differential gene expression, alternative pre-mRNA isoforms, promoter usage and microRNA profiling contribute to define the molecular expression phenotypes implied in the progression of proliferative phenotypes associated to the absence of TIA proteins and prioritize candidates for future study.

overall_design: Each library was run on one RNASeq Multiplex of 76 bp using sequencing from Illumina Genome Analyzer (GAIIx). Three samples were analyzed in this manner, taken from control, TIA1 and TIAR shRNA-depleted HeLa cells.

first: Mario

last: Nuñez

first: Carmen

last: Sánchez-Jiménez

first: José

last: Izquierdo


title: Conservation of mouse-human trans-regulatory circuitry despite high cis-regulatory divergence

first: ENCODE

last: DCC

summary:
The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ~8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ~95% similar with that derived from human TF footprints. However, only ~20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through
evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.

overall_design: We performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ~8.6 million transcription factor (TF) occupancy sites at nucleotide resolution.

first: John

last: Stamatoyannopoulos


title: Transcriptome analysis in mouse late spermatocyte deficient in pachytene piRNAs (Miwi+/- and -/-)

first: Toshiaki

last: Watanabe

overall_design: Late spermatocyte mRNA profiles of Miwi+/- and -/- were analyzed by deep sequencing, in triplicate, using Illumina HiSeq.

first: T

last: Watanable

first: M

last: Zhong

first: E

last: Cheng

first: H

last: Lin


title: High throughput quantitative whole transcriptome analysis of CC10- B4+ and Krt5-CreERT2 - labeled distal lung cells

status: Public on Nov 17 2014

city: PHILADELPHIA

first: Andrew

last: Vaughan

state: United States

address: 3800 Spruce Street

institute: University of Pennsylvania

summary: We used microfluidic single cell RNA-seq on adult isolated CC10-CreERT2 (negative) integrin beta4(pos) cells lung epithelial cells in order to determine the transcriptional profile of this putative progenitor population.

overall_design: CC10-CreERT2 / tdTomato (negative) integrin beta4(pos) cells were isolated by FACS, as were Krt5-CreERT2 / tdTomato (positive) cells.  These cells were pooled and loaded onto the Fluidigm C1 device.

first: A

last: Chapman


title: Change in H3K4me3 in Suz12(Bgal/Bgal) ESCs, and over Spinal Motor Neuron differentiation (ChIP-Seq)

city: Cambridge

first: Laurie

last: Boyer

state: MA

address: 77 Massachusetts Avenue

department: Biology

institute: Massachusetts Institute of Technology

summary: Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated. This data shows the concomitant changes in H3K4me3 levels in these cells.

overall_design: An ESC line mutant in Suz12, a core component of Polycomb Repressive Complex 2, was assessed for H3K4me3 by ChIP-seq, as compared to a wild type ESC line, as well as both lines subjected to in vitro differentiation down the Spinal Motor Neuron pathway.

first: Seraphim

last: Thornton


title: A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential

city: Toronto

first: Jennifer

last: Mitchell

state: Ontario

address: 25 Harbord Street

department: Cell and Systems Biology

country: Canada

institute: University of Toronto

summary: We report the effect on genome-wide gene expression after deletion of an enhancer region downstream of Sox2 in F1 ES cells.  The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency.  Reporter assays reveal novel enhancers, including two enhancers over 100 kb downstream (SRR107 and SRR111) which, through the formation of chromatin loops, localise to the Sox2 promoter in ES cells.  Using CRISPR/Cas9 we deleted a region containing these two enhancers, which we term the Sox2 control region (SCR).  This deletion revealed that the SCR is required for Sox2 transcription in ES cells.  Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies.  Together these data identify a distal control region essential for Sox2 transcription in ES cells.

overall_design: Examination of PolA+ RNA after heterozygous and homozygous enhancer deletion in F1 ES cells (M. musculus129 x M. castaneus).

first: Scott

last: Davidson


title: Defining the nociceptor transcriptome

city: Woodbury

first: Megan

last: Crow

address: 500 Sunnyside Blvd

institute: Cold Spring Harbor Laboratory

summary: Unbiased 'omics' techniques, such as next generation RNA-sequencing, can provide entirely novel insights into biological systems. However, cellular heterogeneity presents a significant barrier to analysis and interpretation of these datasets. The neurons of the dorsal root ganglia (DRG) are an important model for studies of neuronal injury, regeneration and pain. The majority of investigators utilize a dissociated preparation of whole ganglia when studying cellular and molecular function. We demonstrate that the standard methods for producing these preparations gives a 10%-neuronal mixture of cells, with the remainder of cells constituting satellite glia and other non-neuronal cell types. Using a novel application of magnetic purification, we consistently obtain over 95% pure, viable neurons from adult tissue, significantly enriched for small diameter nociceptors expressing the voltage gated ion channel Nav1.8. Using genome-wide RNA-sequencing we compare the currently used (10% neuronal) and pure (95% nociceptor) preparations and find 920 genes enriched. This gives an unprecedented insight into the molecular composition of small nociceptive neurons in the DRG, potentially altering the interpretation of previous studies performed at the tissue level, and indicating a number of novel markers of this widely-studied population of cells. We anticipate that the ease of use, affordability and speed of this technique will see it become widely adopted, delivering a greatly improved capacity to study the roles of nociceptors in health and disease.

overall_design: RNA-Seq was performed for 4 biological replicates from three different groups: intact DRG, acutely dissociated DRG and magnetically-purified DRG neurons. Differential expression was analyzed between acutely dissociated and MACS-dissociated samples to define the 'nociceptor transcriptome'.

first: Stephen

middle: B

last: McMahon


title: Epigenetic and transcriptional regulation of starvation-induced atrophy and autophagy programs by Foxk1 and Sin3A

first: Brian

last: Dynlacht

address: 522 First Avenue

institute: New York University School of Medicine

summary: Autophagy is the primary catabolic process triggered in response to starvation. Although autophagic regulation within the cytosolic compartment is well established, it is becoming clear that nuclear events also regulate the induction or repression of autophagy. Nevertheless, a thorough understanding of the mechanisms by which sequence-specific transcription factors modulate expression of genes required for autophagy is lacking. Here, we identify Foxk proteins (Foxk1 and Foxk2) as transcriptional repressors of autophagy in muscle cells and fibroblasts. Interestingly, Foxk1/2 serve to counter-balance another forkhead transcription factor, Foxo3, which induces an overlapping set of autophagic and atrophic targets in muscle. Foxk1/2 specifically recruits Sin3A-HDAC complexes to restrict acetylation of histone H4 and expression of critical autophagy genes. Remarkably, mTOR promotes the transcriptional activity of Foxk1 by facilitating nuclear entry to specifically limit basal levels of autophagy in nutrient-rich conditions. Our study highlights an ancient, conserved mechanism whereby nutritional status is interpreted by mTOR to restrict autophagy by repressing essential autophagy genes via Foxk-Sin3-mediated transcriptional control.

overall_design: Examination of (1) chromatin binding of Foxk1 and Sin3A in non-starved myoblasts and (2) gene expression profiling upon either starvation or siRNA-mediated depletion of Foxk1 relative to a non-starved control.

first: Christopher

last: Bowman

middle: D


title: Human Nonsense-Mediated RNA Decay Initiates Widely by Endonucleolysis and Targets snoRNA Host Genes

city: Hørsholm

first: Yun

last: Chen

address: Bøge Alle 10

department: Health & Nutrition

country: Denmark

institute: Chr Hansen A/S

summary:
Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogenous substrates has remained unresolved. Here we unambiguously establish that SMG6-catalyzed endonucleolysis is the primary initiating step in human nonsense RNA decay. We also show that both protein-coding and ‘non-coding’ genes hosting snoRNAs in their introns produce considerable amounts of NMD-sensitive splice variants, suggesting that these RNAs are merely by-products of a primary snoRNA production process. Finally, genes encoding multiple snoRNAs generally yield elevated numbers of alternative transcript isoforms, enabling the differential expression of individual snoRNAs. These findings demonstrate a hitherto unappreciated potential for decoupling of the individual expression levels of functional exon- and intron-encoded species from such composite genes.

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This article was corrected in http://genesdev.cshlp.org/content/30/9/1128.short
Completely mapped 5'p-end sequencing data from the corrigendum have been updated in corresponding samples.

overall_design: HEK293 Flp-In T-Rex cells were subjected to siRNA-mediated depletion of XRN1 and co-depletion of XRN1 with either SMG6 or UPF1. All the treated samples together with the controls were subjected to both RNA-seq and 5'-end-seq. RNA-seq was used for detecting NMD isoforms and their expression levels. 5'-end-seq was used for finding NMD decay intermediates (decapped and endocleaved molecules). CAGE was used to distinguish decapped from endocleaved RNA fragments.

first: Søren

last: Lykke-Andersen

first: Torben

last: Jensen

first: Albin

last: Sandelin


title: RNA helicase DDX21 coordinates transcription and noncoding RNA processing of the ribosomal pathway

first: Ryan

last: Flynn

summary: DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense transcriptional status of both RNA Pol I and Pol II to control transcriptional and post-transcriptional steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of protein-coding and noncoding RNAs. Although broad, these molecular interactions, both at the chromatin and at the RNA level, exhibit a remarkable specificity for the ribosomal pathway. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs and, as a functional component of the snoRNA ribonucleoprotein (snoRNP) complex, promotes modification of rRNA. In the nucleoplasm, DDX21 is incorporated into the 7SK snRNP complex, which facilitates DDX21 association with promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of P-TEFb from the 7SK snRNP, enhancing productive Pol II elongation. Altogether, we present a unifying mechanism for the coordinated regulation of ribosomal genes across nuclear compartments, and provide first evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control.

overall_design: Examination of DDX21 chromatin association and DDX21 RNA interacting partners in HEK293 cells

first: Eliezer

last: Calo

first: Howard

last: Chang

first: Joanna

last: Wysocka


title: In vivo activation of a conserved microRNA program induces robust mammalian heart regeneration

city: La Jolla

last: Benner

address: 9500 Gilman Dr. MC 0640

institute: University of California, San Diego (UCSD)

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

sample_organism:
Danio rerio
Rattus norvegicus

platform_organism:
Rattus norvegicus
synthetic construct

overall_design: Refer to individual Series


title: Genome-wide binding patterns of group B Sox proteins in four Drosophila species

city: Basel

first: Sarah

last: Carl

address: Maulbeerstrasse 66

department: Epigenetics

country: Switzerland

institute: Friedrich Miescher Institute

summary: We used DamID-seq to analyze the genome-wide binding patterns of the group B Sox proteins Dichaete and SoxNeuro in four species of Drosophila: D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura. Both binding site turnover between species and a comparison of the binding properties of the two partially-redundant transcription factors were analyzed. We found that, despite widespread turnover, genomic intervals that are commonly bound by both Dichaete and SoxNeuro are highly conserved in Drosophila.

sample_organism:
Drosophila melanogaster
Drosophila pseudoobscura
Drosophila simulans
Drosophila yakuba

overall_design: DamID for Dichaete (Dichaete-Dam) was performed in D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura, while DamID for SoxNeuro (SoxN-Dam) was performed in D. melanogaster and D. simulans. The control experiment, Dam-only, was performed in all species. Three biological replicates were sequenced for each condition in each species.

first: Steve

last: Russell


title: Transcriptomic analysis of  fungus Penicillium oxalicum and its laeA and creA deletion strains

city: Jinan

first: Yuqi

last: Qin

address: 27 Shanda Nan Lu

institute: Shandong University

summary: The goals of this study are to compare differential gene expressions for Penicillium oxalicum wild type strain (WT), laeA knockout strain (ΔlaeA), creA knockout strain (ΔcreA), and double genes knockout strain (ΔlaeAΔcreA). The deletion of laeA downregulated genes involved in oxidation- reduction process, alkaloid metabolic process, and transmembrane transport. We find the expression levels of seven secondary metabolism gene clusters (totally 28 clusters) were silenced inΔlaeA. The deletion of creA upregulated genes involved in hydrolase activity, acting on glycosyl bonds. Many genes involved in conidiation were drastically regulated inΔlaeAΔcreA. This study provides the information that combined laeA and creA function are required in conidiation and hydrolase activity of P. oxalicum.

overall_design: Examination of differential gene expressions by digital gene expression tag profiling in Penicillium oxalicum wild type strain and three mutant strains (laeA knockout strain, creA knockout strain and double genes knockout strain). qRT–PCR validation was performed using SYBR Green assays.

first: Yinbo

last: Qu


title: PRC2 coordinates lineage fidelity and DNA methylation during ESC differentiation (RRBS)

first: Laurie

last: Boyer

summary: Polycomb Repressive Complex 2 (PRC2) catalyzes histone H3 lysine 27 tri-methylation, an epigenetic modification associated with gene repression. H3K27me3 is enriched at the promoters of a large cohort of developmental genes in embryonic stem cells (ESCs). Loss of H3K27me3 leads to a failure of ESCs to properly differentiate, which presents a major roadblock for dissecting the precise roles of PRC2 activity during lineage commitment. While recent studies suggest that loss of H3K27me3 leads to changes in DNA methylation in ESCs, how these two pathways coordinate to regulate gene expression programs during lineage commitment is poorly understood. Here, we analyzed gene expression and DNA methylation levels in several PRC2 mutant ESC lines that maintain varying levels of H3K27me3. We found that maintenance of intermediate levels of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to spinal motor neurons (SMNs). However, genes that function to specify other lineages failed to be repressed, suggesting that PRC2 activity is necessary for lineage fidelity. We also found that H3K27me3 is antagonistic to DNA methylation in cis. Furthermore, loss of H3K27me3 leads to a gain in promoter DNA methylation in developmental genes in ESCs and in lineage genes during differentiation. Thus, our data suggest a role for PRC2 in coordinating dynamic gene repression while protecting against inappropriate promoter DNA methylation during differentiation.

overall_design: Embryonic Stem Cell (ESC)  lines mutant for PRC2 core components Suz12 (Suz12GT and Suz12delta) and Eed (Eednull) were subjected to in vitro directed differentiation down the spinal motor neuron lineage. ESCs and day 5 differentiated cells from the three mutant lines and wild-type were used for Reduced Representation Bisulfite Sequencing (RRBS).

first: Seraphim

last: Thornton


title: PRC2 coordinates lineage fidelity and DNA methylation during ESC differentiation (ChIP-Seq)

overall_design: Embryonic Stem Cell (ESC)  lines mutant for PRC2 core components Suz12 (Suz12GT and Suz12delta) and Eed (Eednull) were subjected to in vitro directed differentiation down the spinal motor neuron lineage. ESCs and day 5 differentiated cells from the three mutant lines and wild-type were used for H3K27me3 ChIP-seq.


title: Selective demethylation and altered gene expression are associated with ICF Syndrome in human induced pluripotent stem cells and mesenchymal stem cells

city: Los Angeles

first: Kevin

last: Huang

address: Gonda BLDG Rm. 6554, P.O.Box 957088

department: Human Genetics

institute: UCLA

summary: Immunodeficiency, Centromeric Instability, and Facial Anomalies Type I (ICF1) Syndrome is a rare genetic disease caused by mutations in DNMT3B, a de novo DNA methyltransferase. However, the molecular basis of how DNMT3B-deficiency leads to ICF1 pathogenesis is unclear. Induced pluripotent stem cell (iPSC) technology facilitates the study of early human developmental diseases via facile in vitro paradigms. Here, we generate iPSCs from ICF Type 1 Syndrome patient fibroblasts followed by directed differentiation of ICF1-iPSCs to mesenchymal stem cells (MSCs). By performing genome-scale bisulfite sequencing, we find that DNMT3B-deficient iPSCs exhibit global loss of non-CG methylation and select CG hypomethylation at gene promoters and enhancers. Further unbiased scanning of ICF1 iPSC methylomes also identifies large megabase regions of CG hypomethylation typically localized in centromeric and subtelomeric regions.  RNA sequencing of ICF1 and control iPSCs reveals abnormal gene expression in ICF1 iPSCs relevant to ICF Syndrome phenotypes, some directly associated with promoter or enhancer hypomethylation. Upon differentiation of ICF1 iPSCs to mesenchymal stem cells (MSCs), we find virtually all CG hypomethylated regions remained hypomethylated when compared to either wild-type iPSC-derived MSCs or primary bone-marrow MSCs. Collectively, our results show specific methylome and transcriptome defects in both ICF1-iPSCs and differentiated somatic cell lineages, providing a valuable stem cell system for further in vitro study of the molecular pathogenesis of ICF1 Syndrome.

overall_design: MethylC-seq and RNA-Seq in ICF Syndrome patient fibroblast derived induced pluripotent stem cells.

first: Guoping

last: Fan


title: PRC2 coordinates lineage fidelity and DNA methylation during ESC differentiation


title: PRC2 coordinates lineage fidelity and DNA methylation during ESC differentiation (RNA-Seq)

overall_design: Embryonic Stem Cell (ESC)  lines mutant for PRC2 core components Suz12 (Suz12GT and Suz12delta) and Eed (Eednull) were subjected to in vitro directed differentiation down the spinal motor neuron lineage. ESCs and day 5 differentiated cells from the three mutant lines and wild-type were used for RNA-seq.


title: The genomic landscape of nasopharyngeal carcinoma

city: Singapore

first: Phillip

last: Koeffler

address: 14 Medical Drive

country: Singapore

institute: Cancer Science Institute

summary: Nasopharyngeal carcinoma (NPC) has extremely skewed ethnic and geographic distributions, is poorly understood at the genetic level and is in need of effective therapeutic approaches. Here we determined the mutational landscape of 128 cases with NPC using whole-exome and targeted deep sequencing, as well as SNP array analysis. These approaches revealed a distinct mutational signature and nine significantly mutated genes, many of which have not been implicated previously in NPC. Notably, integrated analysis showed enrichment of genetic lesions affecting several important cellular processes and pathways, including chromatin modification, ERBB-PI3K signaling and autophagy machinery. Further functional studies suggested the biological relevance of these lesions to the NPC malignant phenotype. In addition, we uncovered a number of new druggable candidates because of their genomic alterations. Together our study provides a molecular basis for a comprehensive understanding of, and exploring new therapies for, NPC

overall_design: Examination of mRNA expression levels in 4 individuals dignosed for Nasopharyngeal Carcinoma using RNA sequencing.

first: De-Chen

last: Lin

first: H

middle: P


title: NRASG12V mediates leukemia self renewal [RNA-Seq]

status: Public on Nov 16 2014

city: Minneapolis

first: Rebecca

last: LaRue

state: MN

address: 117 Pleasant St SE

department: Department of Lab medicine and Pathology

institute: University of Minnesota

summary: Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific function of these pathways in AML is unclear. To elucidate the downstream functions of activated NRAS in AML, we employed a murine model of AML harboring Mll-AF9 and NRASG12V. We found that NRASG12V enforced leukemia self-renewal gene expression signatures and was required to maintain an MLL-AF9 and MYB-dependent gene expression program. In a multiplexed analysis of RAS-dependent signaling intermediates, the leukemia stem cell compartment was preferentially sensitive to RAS withdrawal. Use of RAS-pathway inhibitors showed that NRASG12V maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies.

overall_design: Mice harboring NRASG12V/Mll-AF9 AML were treated with doxycyline to abolish NRASG12V expression.  Leukemia samples were harvested at 24 hour intervals after doxycyline treatment.

first: Zohar

last: Sachs

first: Hanh

middle: T

last: Nguyen

first: Karen

first: Nurul

last: Hassan

first: Ernesto

last: Diaz-Flores

first: Susan

last: Rathe

first: Aaron

last: Sarver

first: Sean

middle: C

last: Bendall

first: Ngoc

last: Ha

first: Miechaleen

last: Diers

first: Gary

last: Nolan

last: Shannon

first: David

last: Largaespada


title: NRASG12V mediates self renewal in AML [RNA-Seq]

overall_design: Primary NRASG12V-Mll-AF9 AML cells were treated in vitro for 24 hours with Ras-pathway inhibitors.  RNA was extracted from these cells and submitted for RNA sequencing.

middle: S

first: Kevin


title: NRASG12V oncogene mediates self-renewal in acute myelogenous leukemia


title: HNF4α is a therapeutic target that links AMPK to WNT signaling in early-stage gastric cancer

status: Public on Nov 15 2014

city: Houston

first: Han

last: Liang

state: TX

address: 1400 Pressler Street

department: Bioinformatics and Computational Biology

institute: The University of Texas MD Anderson Cancer Cencer

summary:
Background Worldwide, gastric cancer is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects.
Objective
To determine oncogenic mechanisms and novel therapeutic targets specific for gastric cancer by identifying commonly dys-regulated genes from the tumors of both Asian-Pacific and Caucasian patients.
Design
We generated transcriptomic profiles of 22 Caucasian gastric cancer tumors and their matched non-cancerous samples, and performed an integrative analysis across different gastric cancer gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signaling pathways by RNAi and/or pharmacologic inhibition.
Results
We found that HNF4α upregulation was a key signaling event in gastric tumors from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumor cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signaling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest, and tumor growth inhibition. HNF4α also regulated WNT signaling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumors.
Conclusions
Our results indicate that HNF4α is a targetable oncoprotein in gastric cancer, is regulated by AMPK signaling through AMPKα, and resides upstream of WNT signaling. HNF4α may regulate “metabolic switch” characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signaling cascade represents a potentially targetable pathway for drug development.

overall_design: Integrative analysis of Caucasian and Asian-Pacific gastric tumor expression datasets (including newly generated transcriptomic profiling of 22 tumors in this study) revealed a relatively small common sets of highly overexpressed genes.

first: Hae

last: Chang

first: Seungyoon

last: Nam

first: Jyeong-Cherl

last: Kook

first: Kyung-Tae

last: Kim

first: Xiuping

last: Liu

first: Hui

last: Yao

last: Jung

first: Robert

last: Lemos

first: Hye

last: Seo

first: Hee

last: Park

first: Youme

last: Gim

first: Dongwan

last: Hong

first: Iksoo

last: Huh

first: Young-Woo

first: Dongfeng

last: Tan

first: Chang-Gong

first: Garth

last: Powis

first: Taesung

first: Yon


title: Transcriptional and epigenetic program changes of induced beta cells over time [RRBS-seq]

first: Kendell

last: Clement

address: 7 Divinity Ave.

institute: Harvard University

summary: Direct lineage conversion is a promising approach to generate therapeutically important cell types for disease modeling and tissue repair.  However, it is often unclear whether lineage-reprogrammed cells remain stable long-term and whether the properties of the reprogrammed cells evolve over time.  Here, using an improved method of converting pancreatic acinar cells to beta-cells, we show that induced beta-cells persist in the adult pancreas for up to 14 months and form islet-like structures.  Detailed analyses of induced cells over 7 months reveal that global DNA methylation changes occur rapidly whereas transcription network remodeling evolves over two months to resemble that of endogenous beta-cells and then stabilizes thereafter.  Progressive gain of beta-cell function by converted cells during the 7 month period coincides with both transcriptional changes and the formation of islet-like structures. These studies demonstrate the ability of lineage-reprogrammed cells to achieve a stable state and identify key cellular and molecular milestones during their long-term evolution.

overall_design: Acinar cells and beta cells were collected as control, as well as induced beta cell samples at day 10, day 30, day 60, and 7 months

first: Weida

last: Li

first: Jiang

last: Zhu

first: Yingying

last: Zhang

first: Qiao

last: Zhou


title: Genome-wide profiling of Tbx6myc binding during zebrafish somitogenesis

city: Coventry

first: Andrew

middle: Christopher

last: Nelson

address: Gibbet Hill Campus

department: School of Life Sciences

institute: University of Warwick

summary: Identification of Tbx6 genomic binding sites during zebrafish somitogenesis in order to identify Tbx6 targets genes

overall_design: Duplicate anti-myc ChIP samples with associated input for heat shocked Tg(hsp70l:tbx6Myc)v8 embryos, plus single paired anti-myc ChIP and input in wild type embryos - 3x ChIP samples; 3x input samples

first: Stefanie

middle: E

last: Windner

first: Chantal

last: Ferguson

first: Rosemarie

last: Doris

last: Oates

first: Fiona

last: Wardle

first: Stephen

last: Devoto


title: Dicer WT/KO MSC RNA-Seq [polyA RNA]

first: Sara

middle: J

last: Gosline

address: 77 Massachusetts Ave

department: Biological Engineering

institute: MIT

summary:
RNA-Seq performed on Dicer KO and WT murine mesenchymal stem cells
MicroRNAs (miRNAs) are small non-coding RNAs that regulates development and disease but induce only moderate repression of directs mRNA targets, suggesting that they coordinate with other modes ofs cellular regulation to effect large changes in gene expression. Ins this work we decouple direct effects of global miRNA loss froms transcriptional changes downstream in a pair of isogenic murines fibroblast cell lines with and without Dicer expression. Wes demonstrate how effects on direct miRNA targets are amplified bys transcription machinery through the construction of a network models that identifies specific transcription factors that cause changes ins mRNA expression upon Dicer loss. Through transcription factors over-expression, we delineate miRNA-mediated transcriptional programss and identify miRNA-mediated coherent and incoherent feed-forwards loops, suggesting a functional role of the interaction between miRNAss and transcription factors.   In total, our results indicate thats miRNAs tightly control transcription factors within a denses interconnected network to modulate gene expression.

overall_design: Examination of mature mRNA expression changes in adult mesenchymal stem cells (immortalized monoclonal lines of murine MSCs) with and without Dicer (WT: Dicer f/f, KO: Dicer -/-).


title: An Endotoxin Tolerance Signature Predicts Sepsis and Organ Dysfunction at Initial Clinical Presentation

status: Public on Nov 14 2014

city: Camperdown

first: David

middle: Graeme

last: Hancock

state: NSW

address: Building 93, RPAH, Missenden Road

country: Australia

institute: Centenary Institute

summary:
Background: Sepsis involves aberrant immune responses to infection, but the exact nature of this immune dysfunction remains poorly defined. Bacterial endotoxins like lipopolysaccharide (LPS) are potent inducers of inflammation, which has been associated with the pathophysiology of sepsis, but repeated exposure can also induce a suppressive effect known as endotoxin tolerance or cellular reprogramming. It has been proposed that endotoxin tolerance might be associated with the immunosuppressive state that was primarily observed during late-stage sepsis. However, this relationship remains poorly characterised. Here we clarify the underlying mechanisms and timing of immune dysfunction in sepsis.
Methods: We defined a gene expression signature characteristic of endotoxin tolerance. Gene-set test approaches were used to correlate this signature with early sepsis, both newly and retrospectively analysing microarrays from 593 patients in 11 cohorts. Then we recruited a unique cohort of possible sepsis patients at first clinical presentation in an independent blinded controlled observational study to determine whether this signature was associated with the development of confirmed sepsis and organ dysfunction.
Findings: All sepsis patients presented an expression profile strongly associated with the endotoxin tolerance signature (p < 0.01; AUC 96.1%). Importantly, this signature further differentiated between suspected sepsis patients who did, or did not, go on to develop confirmed sepsis, and predicted the development of organ dysfunction.
Interpretation: Our data support an updated model of sepsis pathogenesis in which endotoxin tolerance-mediated immune dysfunction (cellular reprogramming) is present throughout the clinical course of disease and related to disease severity. Thus endotoxin tolerance might offer new insights guiding the development of new therapies and diagnostics for early sepsis.

overall_design: For the RNA-Seq study reported here, 73 patients were recruited with deferred consent at the time of first examination in an emergency ward based on the opinion of physicians that there was a potential for the patient's condition to develop into sepsis. These were retrospectively divided into groups based on clinical features and compared to 11 non-urgent surgical controls.

first: Olga

middle: M

last: Pena

middle: G

first: Ngan

middle: H

last: Lyle

first: Adam

last: Linder

first: James

middle: A

last: Russell

first: Jianguo

last: Xia

first: Christopher

last: Fjell

first: John

last: Boyd

first: Robert


title: Leukemia stem cell-enriched population expresses self-renewal gene-expression signature [RNA-Seq]

first: Rebecca

last: LaRue

overall_design: Primary leukemia cells harvested from spleens were sorted into immunophenotypic subpopulations (Mac-1High, Mac-1LowKit–Sca-1–, Mac-1LowKit+Sca-1–, and Mac-1LowKit+Sca-1+). RNA was extracted from this subpopulations of cells and submitted for RNA sequencing.

first: Zohar

last: Sachs

first: Hanh

last: Nguyen

first: Karen

first: Nurul

last: Hassan

first: Ernesto

last: Diaz-Flores

first: Susan

middle: K

last: Rathe

first: Aaron

middle: L

last: Sarver

first: Sean

last: Bendall

first: Ngoc

last: Ha

first: Miechaleen

last: Diers

first: Gary

last: Nolan

first: Kevin

last: Shannon

last: Largaespada


title: An Rtf2 domain-containing protein influences pre-mRNA splicing and is essential for embryonic development in Arabidopsis thaliana

city: Cold Spring Harbor

first: Wen-Wei

last: Liao

address: 1 Bungtown Road

summary:
Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-mRNA splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene. In addition to the known spliceosomal component PRP8, the screen retrieved a previously uncharacterized protein containing a Replication termination factor2 (Rtf2) domain defined by a C2HC2 zinc finger. The Rtf2 protein was discovered in fission yeast, where it stabilizes paused DNA replication forks by an unknown mechanism. When homozygous, a null mutation in Arabidopsis RTF2 (AtRTF2) is embryo-lethal, indicating that it encodes an essential protein. As revealed by quantitative RT-PCR, impaired expression of GFP in atrtf2 and prp8 mutants is attributable to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA-seq demonstrated that 12% of total introns display a significant degree of retention in atrtf2 mutants. Intron-retaining transcripts are enriched from genes encoding proteins involved in signaling pathways and membrane transport. Affinity purification of AtRTF2 followed by mass spectrometry identified several known and predicted splicing proteins. In a yeast two-hybrid screen, AtRTF2 interacted with Exo70B1, a peripheral subunit of the exocyst, which is involved in vesicle trafficking. Considering these results and previous suggestions that Rtf2 constitutes an ubiquitin-related domain, we discuss possible roles of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing and membrane signaling to the spliceosome.
Rtf2 is SDR1 (= AtRTF2) and was discovered in a genetic suppressor screen using the dms4 mutant. DMS4 was described in Kanno et al (2010) EMBO Rep. 11:65-71.

overall_design: Examination of whole-genome DNA methylation status in transgenic Arabidopsis plants

first: Taku

last: Sasaki

first: Tatsuo

last: Kanno

first: Shih-Chieh

last: Liang

first: Pao-Yang

last: Chen

first: Wen-Dar

last: Lin

first: Antonius

last: Matzke

first: Marjori


title: Transcriptome-wide modulation of splicing by the exon junction complex

city: PARIS

first: Herve

last: LE HIR

address: 46 rue d'Ulm

department: Functional Genomics

country: France

institute: IBENS

summary: We report that knockdown of EJC core proteins, eIF4A3, Y14, Magoh, causes a transcript-wide changes in alternative splicing, as well as some transcriptional changes. These changes are specific to EJC core proteins, and KD of UPF1 protein caused different sets of alterantive splicing changes. These changes are linked to the rate of transcription.

overall_design: Examination of 4 different knockdown, as well as GFP knockdown in HeLa cells, 2 replicates each condition.

first: Zhen

last: Wang

first: Valentine

last: Murigneux

last: Le Hir


title: Genome-wide analysis of the RNA polyemerase I trascription factor UBF1/2 binding in mouse and human cell lines.

city: East Melbourne

first: Elaine

last: Sanij

state: VIC

address: 1 St Andrews Place

department: Research

institute: Peter MacCallum Cancer Centre

summary: We report ChIP-Seq analysis of the RNA polyemerase I trascription factor UBF1/2 in NIH3T3, HMEC and HMLER cell lines.

overall_design:
NIH3T3* samples:
We correlated UBF1/2 binding across the genome with that of RNA polemerase I (Pol I), RNA polemerase II (Pol II) and chromatin states in NIH3T3 cells
We perfromed ChIP-seq of UBF1/2 (2 biological replicates), Pol I (POLR1A/RPA194), Pol II, H3K9me3, H3K4Me3, H3K9ac, H4 hyperacetylation and genomic DNA input as reference.

HMEC* samples:
We report ChIP-Seq analysis of UBF1/2 and Pol I (POLR1A/RPA194) in human mammary epithelial cells (HMEC). In addition we include UBF1/2 ChIP-seq in the tumourigenic epithelial cell line HMLER.  In this study, we compared UBF1/2 binding in HMEC to UBF1/2 binding in in the tumorigenic HMLER cells, an isogenic HMEC-derived cell line expressing the SV40 large-T, TERT, and an oncogenic allele of the HRAS gene (expressing HRASV12G) that forms tumors in nude mice (Elenbaas et al., Gene & Development 2001)
We sequenced UBF1/2 ChIP and Pol I(POLR1A/RPA194) ChIP and gDNA input from HMEC. We sequenced UBF1/2 ChIP and gDNA input from HMLER

first: Ross

last: Hannan


title: Role of de novo DNA methyltransferases in hematopoietic stem cells (ChIP-Seq)

first: deqiang

last: sun

address: 2121 W Holcombe Blvd

institute: Texas A&M University

summary: We investigate the dynamics for Histone marks H3K4me3 and H3K27me3 during Dnmt3a and Dnmt3b knockout in mouse hematopoietic stem cells. The term dko represents double knockout of both Dnmt3a and Dnmt3b, while the term sko denotes single knockout of Dnmt3a. The Wildtype profiles were generated in study GSE47765.

overall_design: Mouse hematopoietic stem cell histone methylation profiles of sko and dko mice were generated generated by deep sequencing, in duplicate, using Illumina Hiseq 2000

first: Deqiang

last: Sun


title: Human resistin alters lung mRNA expression from helminth-infected lungs.

city: Riverside

first: Meera

last: Nair

state: California

address: 301 SOM Research Building, 900 University Ave.

department: Biomedical Sciences Department

institute: University of California, Riverside

summary:
Goal: To examine the effects of human resistin during helminth infection.
Methodology: To examine the function of human resistin (hResistin), we utilized transgenic mice expressing the human resistin gene along with its entire regulatory region (hRetnTg+). Following infection with the helminth Nippostrongylus brasiliensis, whole lung RNA was sequenced in hRetnTg+ mice, control hRetnTg- and naïve mice.
Conclusion: In hRetnTg+ mice, many genes involved in inflammation and the immune system, specifically toll-like receptor signaling and chemokines, are significantly upregulated, suggesting that human resistin promotes TLR signaling and inflammation during helminth infection.

overall_design: Examination of whole lung mRNA from Nippostrongylus brasiliensis-infected lungs at day 7 in mice expressing human resistin

first: Jessica

last: Jang


title: The Parathyroid Hormone-Regulated Transcriptome in Osteocytes: Parallel Actions with 1,25-Dihydroxyvitamin D3 to Oppose Gene Expression Changes During Differentiation and to Promote Mature Cell Function [RNA-Seq]

city: Madison

first: Mark

last: Meyer

state: WI

address: 1415 Linden Dr.

department: Nutritional Sciences

institute: University of Wisconsin-Madison

summary: Although localized to the mineralized matrix of bone, osteocytes are able to respond to systemic factors such as the calciotropic hormones 1,25(OH)2D3 and PTH. In the present studies, we examine the transcriptomic response to PTH in an osteocyte cell model and found that this hormone regulated an extensive panel of genes.  Surprisingly, PTH uniquely modulated two cohorts of genes, one that was expressed and associated with the osteoblast to osteocyte transition and the other a cohort that was expressed only in the mature osteocyte. Interestingly, PTH’s effects were largely to oppose the expression of differentiation-related genes in the former cohort, while potentiating the expression of osteocyte-specific genes in the latter cohort. A comparison of the transcriptional effects of PTH with those obtained previously with 1,25(OH)2D3 revealed a subset of genes that was strongly overlapping.  While 1,25(OH)2D3 potentiated the expression of osteocyte-specific genes similar to that seen with PTH, the overlap between the two hormones was more limited. Additional experiments identified the PKA-activated phospho-CREB (pCREB) cistrome, revealing that while many of the differentiation-related PTH regulated genes were apparent targets of a PKA-mediated signaling pathway, a reduction in pCREB binding at sites associated with osteocyte-specific PTH targets appeared to involve alternative PTH activation pathways.  That pCREB binding activities positioned near important hormone-regulated gene cohorts were localized to control regions of genes was reinforced by the presence of epigenetic enhancer signatures exemplified by unique modifications at histones H3 and H4. These studies suggest that both PTH and 1,25(OH)2D3 may play important and perhaps cooperative roles in limiting osteocyte differentiation from its precursors while simultaneously exerting distinct roles in regulating mature osteocyte function. Our results provide new insight into transcription factor-associated mechanisms through which PTH and 1,25(OH)2D3 regulate a plethora of genes important to the osteoblast/osteocyte lineage.

overall_design:
Fully differentiated IDG-SW3 cells were treated  in biological triplicate with 100nM PTH for 24 hours prior to mRNA isolation and sequencing.  Vehicle treated samples were previously published in GSE54783:
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1323967
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1323968
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1323969

first: Hillary

last: St. John

first: J

middle: W

last: Pike


title: Genome-wide analysis of intracellular notch1(ICN1) targets in gamma delta T cells (gd T cells).

city: Fukuoka city

first: Masataka

last: Nakamura

state: Fukuoka prefecture

address: 3-1-1 Maidashi

department: Medical Institute of Bioregulation

country: Japan

institute: Kyushu University

summary: We performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) for ICN1 in thymic gd T cells from neonates of C57BL/6 mice. Sequence reads for ICN1 and Input were aligned with the mouse reference genome (mm10) by bowtie program (v1.0.0), and peak detection and identification of ICN1 binding sites were obtained with MACS program (v1.4.2). ChIP-seq data revealed that 12%, 6% and 47% of ICN1 binding sites were within -10kb upstream of transcription start site, +10kb downstream of 3'end and the bodies of genes, respectively. The other of ICN1 binding sites was intergenic. We also found that ICN1 directly bound -5.4kb upstream of transcript start site of IL-7 receptor alpha (IL-7Ra) locus, which were estimated as a new promoter region of IL-7Ra. Some of ICN1 binding sites (e.g. TCF1 promoter) were consistent with previous reports, but others (e.g. Nr4a1 promoter) were novel. These suggests that ICN1 was associated with a lot of transcriptional regulation in gd T cells.

overall_design: Examination of ICN1 binding sites in gd T cells

first: Tetsuya

last: Sato

first: Yasuyuki

last: Ohkawa


title: PRMT5 protects genomic integrity during global DNA demethylation in primordial germ cells and preimplantation embryos

city: St Louis

first: Sabine

last: Dietmann

state: MO

address: 4565 McKinley Avenue

institute: Washington University School of Medicine

summary: Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which entails comprehensive erasure of DNA methylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs caused complete male and female sterility, which is preceded by upregulation of LINE1 and IAP transposons and DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation and early embryonic lethality. We detected the PRMT5-catalysed repressive H2A/H4R3me2s modification on LINE1 and IAP in early wildtype PGCs, directly implicating this mark in the maintenance of transposon silencing during DNA hypomethylation. PRMT5 subsequently translocates back to the cytoplasm of PGCs to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA re-methylation. Thus, PRMT5 has a novel direct role in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation

overall_design: PGCs mRNA profiles of embryonic day 11.5 dpc control (Blimp1Cre;Prmt5flox/+) and Prmt5 germ cell knockout (Blimp1Cre;Prmt5 flox/flox) were generated by deep sequencing (SOLiD next generation sequencing).

first: Shinseog

last: Kim


title: Small RNA accumulation in Transposable Element-silent and Transposable Element-active epigenomes

city: St. Louis

first: R. Keith

last: Slotkin

state: Missouri

address: 975 North Warson Road

institute: Donald Danforth Plant Science Center

summary:
Background: The small RNAs that Transposable Elements generate are vastly different when they are transcriptionally silenced compared to when they are transcriptionally activated. We performed the deep sequencing of small RNAs in a number of small RNA biogenesis mutants in both Transposable Element-silenced and Transposable Element-active epigenome backgrounds.
Results: We found that Transposable Elements generate large amounts of 21-22nt siRNAs only when they are transcriptionally active. These 21-22nt siRNAs are incorporated into the AGO6 protein.
Conclusion: Ago6 is the key protein that bridges the post-transcriptional degradation of Transposable Element mRNAs and the establishment of DNA methylation.

overall_design:
Examination of flower bud small RNAs from wild type and 5 single or double mutant combinations, many of which have biological replicates. In addition, IP purification of the AGO6 protein (and mock no-antigen controls) followed by sequencing of the incorporated small RNAs.

Replicate A for Col and ddm1 are submitted in GSE41755

first: R

first: Andrea

last: McCue

first: Kaushik

last: Panda


title: The Parathyroid Hormone-Regulated Transcriptome in Osteocytes: Parallel Actions with 1,25-Dihydroxyvitamin D3 to Oppose Gene Expression Changes During Differentiation and to Promote Mature Cell Function [ChIP-Seq]

middle: B

overall_design: ChIP-seq was performed on MC3T3-E1 cells for Phospho-Creb following 1 hour vehicle or forskolin treatment in biological replicate.  Input sample was previously published in GSE41920 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1027508).


title: Genome wide mapping of AR and its associated factors in prostate cancer

city: Itabashi-ku

first: Ken-ichi

last: Takayama

address: Sakaecho

institute: Tokyo Metropolitan Institute of Gerontology

summary: Prostate cancer is the most common cancer in men and AR downstream signalings promote prostate cancer cell proliferation.We performed ChIP-seq analysis to investigate the role of AR and its associated factors such as coregulators or collaborating factors.In addition, by siRNA mediated knockdown of such factors, changes of AR-binding sites in prostate cancer cells were analyzed.

overall_design: ChIP-sequence analysis of AR and its associated factors in prostate cancer cells

first: Satoshi

last: Inoue


title: Transcriptome profiling of muscle by RNA-Seq reveals significant digital gene expression profiling differences between Angus and Luxi Cattle

city: jinan

first: liu

middle: gui

last: fen

state: shandong

address: sangyuan road 8

institute: Institute of animal science and veterinary medicine Shandong academy of agricultural sciences

summary: The development of massively parallel sequencing technologies enables the sequencing of total cDNA to identify unigene expression and to discover novel regions of transcription. Here, we report the first use of RNA-Seq to find the digital gene expression profiles (DGEs) associated with the growth and development of muscle in both Chinese Luxi and Angus beef cattle. More than 9,243,921 clean reads were found in samples of muscle tissue. We found 232 DGEs between Luxi cattle and Angus cattle (FDR≤0.001 AND |log2Ratio|≥1). Among the DGEs, we determined that 147 genes were down-regulated and 85 genes were up-regulated. GO and Pathway analysis were performed to analyze the biological role of the DGEs and determine their contribution to the differences seen in muscle growth and development between local Chinese Luxi cattle and the introduced Angus cattle. This article suggests that RNA-Seq is a useful tool for predicting differences in gene expression between Luxi and Angus beef cattle; moreover, our result provides unprecedented resolution of mRNAs that are expressed across the two breeds.

overall_design: Three Luxi and three Angus cattle that were eighteen months of age were generated by RNA-Seq

last: guifen

first: wan

last: fachun


title: Genome-wide map of H3K4me3 binding sites by ChIP-seq in human lymphoblastoid cell lines treated with doxorubicin

city: Research Triangle Park

first: Xuting

last: Wang

state: NC

address: 111 TW Alexander Dr

institute: NIEHS/NIH

summary: We have used chromatin immune-precipitation with parallel sequencing (ChIP-Seq) technology to identify genome-wide H3K4me3 binding in human lymphoblastoid cell lines treated with a DNA-damaging chemotherapeutic reagent doxorubicin.

overall_design: ChIP-Seq analysis of H3K4me3 binding sites in human lymphoblastoid cells treated with Doxorubicin or vehicle

first: Dan

last: Su

first: Douglas

last: Bell


title: Establishment of Highly Tumorigenic Human Colorectal Cancer Cell Line (CR4) with Properties of Putative Cancer Stem Cells

city: Stony Brook

first: Yuanhao

last: Zhang

address: AMS Department,Stony Brook University

department: Applied Mathematics and Statistics

institute: Stony Brook University

summary: Colorectal cancer (CRC) has the third highest incidence and mortality rates among the US population. According to the most recent concept of carcinogenesis, human tumors are organized hierarchically, and the top of this hierarchy is occupied by malignant stem cells, or cancer stem cells (CSCs), which possess unlimited self-renewal and tumor-initiating capacities and high resistance to conventional anticancer therapies. To reflect the complexity and diversity of human tumors and to provide clinically and physiologically relevant in vivo and in vitro models, a large banks of well characterized patient-derived low-passage cell lines, and especially CSC-enriched cell lines are urgently needed.

overall_design: Using RNA-Seq, we have performed a functional genomic analysis in tumor-initiating fractions of CR4 (small) cells grown adherent to type I collagen versus grown as 3D spheroids, in comparison to the bulk tumor cells (long and dychotomized cells) grown under standard culture conditions.

first: Ellen

last: Li

first: Galina

middle: I

last: Botchkina

first: Rebecca

last: Rowehl


title: RNA sequencing of the developing zebrafish head

status: Public on Nov 13 2014

city: Boston

first: Michael

middle: E.

last: Talkowski

address: 185 Cambridge Street

department: Center for Human Genetic Research

institute: Massachusetts General Hospital

summary: We sequenced strand-specific mRNA from the heads of 3 groups of wild type zebrafish (Danio rerio) 5 days post fertilization.

overall_design: Examination of the relative expression of genes in the developing zebrafish brain

first: Ian

last: Blumenthal

first: Christelle

last: Golzio

first: Nicholas

last: Katsanis


title: The C. elegans SNAP complex component SNPC-4 coats piRNA domains and globally affects piRNA abundance

city: New Haven

first: Valerie

last: Reinke

state: CT

address: 333 Cedar St

department: Genetics

institute: Yale University School of Medicine

summary: Remarkably, tens of thousands of piwi-interacting RNAs (piRNAs) arise from a minimal number of discrete clustered regions in many organisms. However, it remains unclear if organization into these domains contributes to the highly coordinated, germline-specific expression of these small RNAs. Here, we show that SNPC-4, the DNA-binding subunit of the small nuclear RNA activating protein complex (SNAPc), binds piRNA clusters in a germline-specific manner and is required for global piRNA expression in C. elegans. Within piRNA domains, SNPC-4 not only binds at discrete sites through its established sequence motif, but also exhibits widely distributed binding across the region. Intriguingly, discrete peaks are not found at every piRNA, but instead occur frequently at Pol III-occupied tRNA genes, which have been previously implicated in chromatin organization. Given its unique binding pattern, we suggest that SNPC-4 promotes robust piRNA expression by establishing a permissive chromatin environment at clusters specifically in the germ line.

overall_design:
This data submission includes a total of 11 ChIP-seq samples and 2 RNA-seq samples.
The ChIP-seq samples include six timecourse samples (Emb-YA) and three tissue-specific samples (WA, GL, SO) for one factor called SNPC-4 (previously called GEI-11).  The remaining two ChIP-seq samples are for pol II (AMA-1) and pol III (RPC-1) at one developmental stage.  All of these samples were done in replicate, with inputs, yielding at least four sequencing files per sample.  Some of the ChIP-seq samples already have fastq files submitted to GEO:  (SNPC-4 EMB _GSE48744: GSM1183805, GSM1183806, GSM1183807, GSM1183808), (SNPC-4 L1_GSE48702: GSM1183629, GSM1183630, GSM1183631, GSM1183632), (SNPC-4 L2_GSE37809: GSM928364, GSM928365, GSM928366, GSM928367), (SNPC-4 L3_GSE35276: GSM864851, GSM864852, GSM864853, GSM864854), and (AMA-1_GSE15535: GSM389309, GSM389310, GSM389312, GSM389313).  We are submitting fastq files for the remaining ChIP-seq datasets:  SNPC-4 L4 - 4 files, SNPC-4 YA - 5 files, SNPC-4 WA - 4 files, SNPC-4 GL - 4 files, SNPC-4 SO - 4 files, and RPC-1 WA - 5 files.  Thus we are submitting an additional 26 fastq files.  Additionally, we are submitting processed files for all 11 datasets, consisting of one normalized file for the combined duplicate IP and one normalized file for the input sequence.  In the case of the three tissue-specific samples for SNPC-4, we are submitting an additional processed (but not normalized) file that represents the IP data with input-subtracted.  Thus, we are submitting a total of 25 processed files.  NOTE:  The processed files for the ChIP-seq samples that already have fastq files submitted to GEO (SNPC-4 EMB _GSE48744, etc.) are linked below as supplementary files at the foot of the Series record.
The RNA-seq samples represent one control (control_RNAseq) and one treatment (SNPC-4(RNAi)_RNA-seq)and were not duplicated.  We are submitting one fastq file for each, as well as one processed data file for each, for a total of 2 fastq and 2 processed data files.

first: Dionna

last: Kasper

first: Guilin

first: Kathryn

last: Gardner

first: Timothy

last: Johnstone


title: Cas9 target DNA specificity

city: Stanford

first: Andrew

last: Fire

address: 300 Pasteur Drive Lane building L302

institute: Stanford

summary: To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage.  Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in "seed" sequences matching the proximal 8 bp of the guide.  A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13-18 relative to the PAM).  Strikingly, a subset of variants which broke homology in the intervening region consistently increased the capacity of Cas9 to cleave in extended reactions. Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage.  Taken together, these studies provide both a basis for predicting effective cleavage targets and a basis for potential optimization of guide RNAs to yield efficiency beyond that of the simple perfect-match guides.

overall_design:
118 samples anaylzed. Controls have con in sample name.
To quantitatively measure cleavage efficiency of a single gRNA, we created a population of random variant target sequences to two gRNA targets.  The targets used were "unc-22A", [a sequence from the well-characterized unc-22 gene of Caenorhabditis elegans], and "protospacer 4" (ps4), a previously characterized sequence from a natural spacer from S. pyogenes MGAS10750 . Using custom mixtures of oligonucleotide precursors for each base during chemical synthesis, a set of polymorphic target libraries ('Random Variant Libraries') were designed to have a baseline variation rate at each position. On each side of the gRNA homology and PAM regions, 6 bps of random sequence were added. The first base of intended gRNA homology is designated base 1 . The entire 35 bp random variant library mixture was cloned into a standard plasmid vector (pHRL-TK). Several thousand colonies from plates were washed in pools and prepared by standard plasmid preparation methods.  The complexity of the libraries were estimated based on Illumina sequencing of the uncut libraries and filtering for minimum representation expected from the pooling.  Approximately 1500-3000 unique species were obtained in the unc-22A libraries and 5000 unique sequences in the ps4 library (see Materials and Methods).

To assay cleavage, purified Cas9 was first incubated with gRNA, followed by incubation with the variant library for various time points and under various conditions. DNA template is among the conditions varied in the experiments.  After protein removal, flanking sequences outside of the target region are used for PCR amplification and plasmid cleavage was measured through loss of PCR products that span the region of interest.  A set of perfectly matched targets and highly mutated versions present in the random variant library served as internal positive and negative controls respectively.

A log retention score for each sequence in each experiment was calculated by quantifying the representation of each sequence before and after addition of the Cas9 protein. Two approaches were used for normalization: first we used a population of ps4 targets "spiked" into the library as an uncleaved control, second, we used a population of unc-22A targets with large numbers of variations from the perfect target (between 4 and 7), and hence likely limited if any cleavage.  Equivalent results are obtained with these two normalization approaches (see Computational Methods for details). Retention scores are expressed as the log2 of the normalized ratio, so that a more negative retention score indicates efficient cleavage of substrate while a less negative score indicates less cleavage. Templates which are uncleaved will yield a retention score at or near zero.  Comparisons between multiple experiments indicate strong correlation between independent retention measurements.

GSM1410678-GSM1410761; AF_SOL*.dat' files contain the calculated final retentions for each experiment. Each experiment labeled: “AF_SOL_###_t###”. “AF_SOL_###” corresponds to the experiment run ID and “t###” corresponds to the incubation time of the experiment. For example AF_SOL_513_t360, corresponds to experiment 513 on the protospacer 4 guide and DNA target and the incubation time was 360 mins. The experimental conditions and ID can be found in the associated publication.

GSM1544297-GSM1544332; unc*.dat file is a tab-delimited file of all considered sequences in each experiment. The names of the files and the AF_SOL_# run number can be found in the associated publication (Supplementary Materials) with the details of the conditions. Each filename starts with the type of gRNA used (either unc-22WT or the mutant version unc22C11G). The next number (#min) is indication of the time of incubation for the experiment and this is either followed by #pcr_AF_SOL_# or just AF_SOL_#. If followed by #pcr, that is the indication of the number of PCR cycles used in the experiments. Finally, AF_SOL_# denotes the sequencing run ID number.