title: Genome-wide binding analysis of GATA1fl and GATA1s in G1ME cells.

city: University Park

first: Ross

last: Hardison

state: PA

address: 303 Wartik Lab

institute: Pennsylvania State University

summary: We used chromatin immunoprecipitation sequencing (ChIP-seq) to compare genome-wide binding profiles of GATA1fl and GATA1s in G1ME cells, which are immortalized, developmentally arrested megakaryocyte-erythroid progenitors (MEPs) derived from in vitro differentiation of murine Gata1- ES cells. Although the truncation in GATA1s leaves the DNA binding domain intact, GATA1s fails to broadly occupy erythroid specific regulatory regions. These observations point to lineage specific co-factor associations essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease.

overall_design: We performed ChIP-seq on G1ME cells 42 hours after retroviral transfer of HA-tagged full-length GATA1 (GATA1fl) or GATA1s cDNAs (2 replicates each). At this time point, there was no apparent difference in the cell surface phenotypes between GATA1fl and GATA1s-expressing cells. For each ChIP-seq replicate we obtained a matching input sample (non-ChIP DNA) as a control.

first: Marta

last: Byrska-Bishop

first: Daniel

last: VanDorn

first: Amy

last: Campbell

first: Marisol

last: Betensky

first: Philip

last: Arca

first: Yu

last: Yao

first: Paul

last: Gadue

first: Fernando

last: Costa

first: Richard

last: Nemiroff

first: Gerd

last: Blobel

first: Deborah

last: French

first: Mitchell

middle: J

last: Weiss

first: Stella

middle: T

last: Chou


title: Pluripotent stem cells reveal novel erythroid activities of the GATA1 N-terminus

status: Public on Jan 26 2015

country: USA

type: Genome binding/occupancy profiling by high throughput sequencing

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

Germline GATA1 mutations resulting in the production of an amino-truncated protein termed GATA1s (for “short”) cause congenital hypoplastic anemia. Similar somatic mutations promote transient myeloproliferative disease and acute megakaryoblastic leukemia in trisomy 21 patients. Here we show that induced pluripotent stem cells (iPSCs) from patients with GATA1-truncating mutations exhibit impaired erythroid potential but enhanced megakaryopoiesis and myelopoiesis, faithfully recapitulating the major phenotypes of associated diseases. Similarly, GATA1s promotes megakaryopoiesis but not erythropoiesis in developmentally arrested Gata1- murine megakaryocyte-erythroid progenitors derived from murine embryonic stem cells (ESCs). Transcriptome studies demonstrate a selective deficiency in the ability of GATA1s to activate erythroid-expressed genes within populations of hematopoietic progenitors. Although its DNA binding domain is intact, chromatin immunoprecipitation studies show that GATA1s binding at specific erythroid regulatory regions is impaired, while binding at many non-erythroid sites, including megakaryocytic and myeloid target genes, is normal. These observations point to lineage specific GATA1 co-factor associations essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease. More broadly, our studies underscore the value of ESCs and iPSCs to recapitulate and study disease phenotypes.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Evaluation of covS polymorphisms on group A streptococcal global gene expression

status: Public on Jan 24 2015

city: Houston

first: Samuel

last: Shelburne

state: TX

address: 1515 Holcombe Blvd

department: Infectious Diseases

institute: MD Anderson Cancer Center

type: Expression profiling by high throughput sequencing

summary: We sought to determine how single amino acid changes in the active site of CovS autophosphorylation and subsequent phosphorylation of the response regulator CovR affect the gene expression profile of group A streptococcus

overall_design: There were 6 strains analyzed, each in quadruplicate replicates: 1) wild-type GAS; 2) covS deletion strain; 3) covS with alanine at AA 280; 4) covS with valine at AA 281; 5) covS with valine at AA 284; 6) covS with alanine at AA 457

middle: A

first: Nicola

last: Horstmann

first: Hui


title: Transcriptome of wild-type and G9a mutant upon viral challenge

city: Nijmegen

first: Ronald

last: van Rij

address: Geert Grooteplein 28

country: Netherlands

institute: Radboud Institute for Molecular Life Sciences - Radboud University Medical Center

summary: The aim of the study was to generate transcriptome of wild-type and G9a mutant adult flies (females) 24h post-infection with  Drosophila C Virus (DCV).

overall_design: We generated 8 different data sets. For wild-type controls and G9a mutants, we performed both mock and DCV infection, and collected both whole flies and fat bodies. All flies were 3-5 days old females.

middle: P

first: Sarah

middle: H

last: Merkling


title: Genome-wide map of spliced-leader-led trans-splice sites in Oikopleura dioica

city: Bergen

first: Eric

last: Thompson

address: Thormøhlensgt. 55

country: Norway

institute: Sars International Centre for Marine Molecular Biology

summary: Polycistronic mRNAs transcribed from operons are resolved via the trans-splicing of a spliced leader (SL) RNA. The SL is also frequently trans-spliced to monocistronic transcripts. Using a modified cap analysis of gene expression (CAGE) protocol we mapped sites of SL trans-splicing genome-wide in the marine chordate Oikopleura dioica and find evidence for proposed functions of SL-trans-splicing. A recent hypothesis postulates that operons facilitate recovery from growth arrested states in metazoans. We examined the expression dynamics of operons across the life-cycle of the animal and during growth arrest recovery. We show that operons do not facilitate recovery from growth arrest in O. dioica. We find that operons are enriched in the germline and that trans-spliced transcripts are predominantly maternal., Interestingly, there is a TOP-like motif in the SL sequence, and trans-splicing in TOP mRNAs, indicating that trans-spliced mRNAs are targets for nutrient-dependent translational control in O. dioica.

overall_design: Total RNA from a number of stages across development were pooled and used in a modified DeepCAGE protocol. A custom designed spliced-leader primer (using the SL exon) was used in the 2nd strand synthesis step.

first: Gemma

middle: B

last: Danks

first: Martina

last: Raasholm

first: Coen

last: Campsteijn

first: Abby

middle: M

last: Long

first: J

middle: R

last: Manak

first: Boris

last: Lenhard


title: High throughput sequencing identifies alternative splicing events regulated by PRMT9

status: Public on Jan 22 2015

city: Portland

first: Zheng

last: Xia

state: Oregon

address: 3181 SW Sam Jackson Park Rd

department: Department of Biomedical Engineering

institute: Oregon Health & Science University

summary: The human genome encodes a family of nine protein arginine methyltransferases (PRMT1-9). Different members of this enzyme family catalyze different types of protein methylation at the terminal nitrogen atoms of arginine residues, forming monomethylated arginine (MMA), asymmetrically dimethylated arginine (ADMA) and symmetrically dimethylated arginine (SDMA). The last member of this family, PRMT9, is characterized in detail here. We identify two spliceosome-associated proteins, SAP145 (SF3B2) and SAP49 (SF3B4), as PRMT9 binding partners, linking PRMT9 to U2snRNP maturation.  We show that SAP145 is methylated by PRMT9 at arginine 508 (R508). Amino acid analysis and a methyl-specific antibody revealed the formation of MMA and SDMA, and PRMT9 thus joins PRMT5 as the only mammalian enzymes that can deposit the SDMA mark. Methylation of the SAP145R508 generates a binding site for the Tudor domain of SMN, and RNA-seq analysis reveals gross splicing changes when PRMT9 levels are attenuated. These studies identify PRMT9 as a non-histone methyltransferase that primes the U2snRNP for interaction with SMN.

overall_design: RNA sequencing was carried out using RNA samples from two biological replicates of control and PRMT9 knockdown HeLa cells.

first: Yanzhong

last: Yang

first: Wei

last: Li

first: Mark

last: Bedford


title: Transcriptional Analysis of Deinococcus radiodurans for Novel sRNAs expression that are Differentially Expressed under Ionizing Radiation

city: Austin

first: Chen-Hsun

last: Tsai

state: Texas

address: Room 3.416, 200 E. Dean Keeton St. Stop C0400

department: McKetta Department of Chemical Engineering

institute: Univ. of Texas at Austin

type: Non-coding RNA profiling by high throughput sequencing

summary: In this study, we identified potential sRNA candidates in D. radiodurans by a whole transcriptome deep sequencing analysis, and confirm the expression of 31 sRNAs in D. radiodurans with Northern blotting analysis and RT-PCR. A total of 8 confirmed sRNAs showed differential expression during recovery after acute ionizing radiation (15 kGy).

sample_organism: Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539

overall_design: Two samples (sham and 15kGy irradiated) were sequenced.

first: Lydia

last: Contreras


title: Evolutionary consequences of DNA methylation on the GC content in vertebrate genomes

status: Public on Jan 23 2015

city: Uppsala

first: Hans

last: Ellegren

address: Norbyvägen 18D

department: Ecology and Genetics

country: Sweden

institute: Uppsala University

type: Methylation profiling by high throughput sequencing

summary: The genomes of many vertebrates show a characteristic variation in GC content. To explain its origin and evolution mainly three mechanisms have been proposed, selection for GC content, mutation bias and GC-biased gene conversion. At present the mechanism of GC-biased gene conversion, i.e. short-scale, unidirectional exchanges between homologous chromosomes in the neighborhood of recombination-initiating double-strand breaks in favor for GC nucleotides, is the most widely accepted hypothesis. We here suggest that DNA methylation also plays an important role in the evolution of GC content in vertebrate genomes. To test this hypothesis we investigated one mammalian (human; GSE30340) and one avian (chicken) genome. We used bisulfite sequencing to generate a whole-genome methylation map of chicken sperm. Human processed data files (spermdonor1, #reads>=1) were downloaded from the NGSmethDB database (http://bioinfo2.ugr.es/NGSmethDB/database.php). Inclusion of these methylation maps into a model of GC content evolution provided significant support for the impact of DNA methylation on the local equilibrium GC content. Moreover, two different estimates of equilibrium GC content, one which neglects and one which incorporates the impact of DNA methylation and the concomitant CpG hypermutability, give estimates that differ about 15% in both genomes, arguing for a strong impact of DNA methylation on the evolution of GC content. Thus, our results put forward that previous estimates of equilibrium GC content, which neglect the hypermutability of CpG dinucleotides, need to be reevaluated.

overall_design: Genomic DNA from chicken mature sperm was isolated, bisulfite converted and sequenced on a Illumina HiSeq instrument

first: Carina

middle: F

last: Mugal

first: Peter

last: Arndt

first: Lena

last: Holm


title: Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation

city: Research Triangle Park

first: Scott

middle: Alexander

last: Lujan

state: NC

address: 101 T.W. Alexander Drive

department: LMG/LSB

institute: NIEHS

summary: Multiple DNA polymerases are needed to replicate genetic information. Here we describe the use of ribonucleotide incorporation as a biomarker of replication enzymology in vivo. We find that ribonucleotides are incorporated into the yeast nuclear genome in replicase specific and strand-specific patterns that identify replication origins and where polymerase switching occurs. Ribonucleotide density varies across the genome as a function of the replicase, base, local sequence and proximity to nucleosomes and transcription start sites. Ribonucleotides are present in one strand at high densityat mitochondrial replication origins, implying unidirectional replication of a circular genome. The evolutionary conservation of the enzymes that incorporate and process ribonucleotides in DNA suggests that the use of ribonucleotides as biomarkers of DNA synthesis in cells will have widespread applicability.

overall_design: Mapping genomic ribonucleotides in 14 Saccharomyces cerevisiae strains (seven DNA polymerase backgrounds, with or without RNH201), via HydEn-seq (end sequencing of genomic fragments generated by alkaline hydrolysis).

first: Anders

last: Clausen

first: Adam

last: Burkholder

first: Clinton

middle: D

last: Orebaugh

first: Jessica

middle: S

last: Williams

first: Maryam

first: Ewa

last: Malc

first: Piotr

last: Mieczkowski

first: David

middle: C

last: Fargo

first: Duncan

last: Smith

first: Thomas

last: Kunkel


title: Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation

city: Basel

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

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

DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined chromosomal binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects chromosomal binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.


title: Jarid2 methylation is required for PRC2 functioning during ES cells differentiation

city: Paris Cedex 05

first: Nicolas

last: Servant

address: 26 rue d'ulm

country: France

institute: Institut Curie

summary: We report that Jarid2 is methylated at K116 by the polycomb complex PRC2. We analyzed Jarid2 versus Methylated Jarid2 genomic localization by ChIP-seq. We also study H3K27 enrichment in Jarid2 knock out ES cells and in cells rescued for Jarid2 or a mutant that cannot be methylated. ChIP-seq for H3K27me3 was perfomed in undiffrentiated ES cells as well as after 8 days of diffrentiation toward Embroyid Bodies (Hanging Drop method).

overall_design: Experiment 1: Jarid2 vs Jarid2me ChIP-seq in undifferentiated mouse ES cells (E14). Experiment 2: H3K27me3 ChIP-seq in Jarid2 -/- ES cells (peng, 2009, Cell), or the same cells rescue with a BAC expressing JARID2 or a mutant of K116 to A. H3K27me3 Chip-seq were perfomed in undifferentiated ES cells and in EB after 8 days of differentiation (hanging Drop method).

first: Aurélie

last: Teissandier

first: Raphaël

last: Margueron


title: Effect of Rps5 heterozygous deletion on embryonic stem cells transcriptome

city: Montreal

first: Guy

last: Sauvageau

state: QC

address: 2950, Chemin de Polytechnique

country: Canada

institute: Institute for Research in Immunology and Cancer

summary: Using wild-type and Rps5 heterozygous embryonic stem cells, we isolated RNA from polyribosomal fractions in order to get insights into transcriptional and translational defects of such deletion.

overall_design: Input, monosomes and polysomes extracted RNA samples from wild-type and Rps5 heterozygous clones (undifferentiated and differentiated, total number of samples = 12), were subjected to sequencing.

first: Simon

last: Fortier


title: Analysis of allele specific expression and its chromatin state to identify genes that are escaping X chromosome inactivation

city: Paris

first: Ronan

last: Chaligné

address: 26, Rue d'Ulm

summary: Disappearance of the Barr body has long been considered a hallmark of cancer, although whether this corresponds to epigenetic instability and transcriptional reactivation, or to genetic loss, has remained unclear. Here we show that in breast cancer cell lines as well as primary breast tumors, the inactive X chromosome frequently displays a highly abnormal 3D nuclear organization, with global perturbations in its characteristic heterochromatic state, including apparent gain of euchromatic marks and lessening of repressive marks such as H3K27me3 and promoter DNA methylation. Genome-wide profiling of chromatin and transcription reveal modified epigenomic landscapes in cancer cells accompanied by a significant degree of X-linked gene reactivation, affecting genes previously implicated in cancer, including the histone deacetylase, HDAC8 and transducin (Beta)-Like 1X-Linked, TBL1X. We provide proof of principle that epigenetic deregulation can indeed perturb the dosage of some X-linked factors and demonstrate that many of these genes are reactivated in primary breast tumors. Our study establishes that the inactive X is subject to epigenetic erosion in a cancer context and sets the stage for the use of chromatin marks and X- chromosome genes as potential biomarkers to assess epigenetic changes in cancer.

overall_design: Examination of allele specific expression of genes in chrX using histone marks, transcriptome, exome and SNP data on two normal cell-line and three cancer cell-line.

last: Chaligne

first: Tatiana

last: Popova

first: Marco-Antonio

last: Mendoza-Parra

first: Mohamed-Ashick

last: Saleem

last: Gentien

first: Kristen

last: Ban

first: Tristan

last: Piolot

first: Olivier

last: Leroy

first: Odette

last: Mariani

first: Hinrich

last: Gronemeyer

first: Anne

last: Vincent-Salomon

first: Marc-Henri

last: Stern

first: Edith

last: Heard


title: Defining the transcriptome landscape of Candida glabrata during pH change and nitrosative stress by RNA-Seq

status: Public on Jan 27 2015

city: Jena

first: Jörg

last: Linde

address: Naumburger Str. 96 a

department: Institute for Bacterial Infections and Zoonoses

country: Germany

institute: Federal Research Institute for Animal Health - Friedrich-Loeffler-Institute

summary: The yeast Candida glabrata is one of the most important human fungal pathogens and has emerged as a leading cause of nosocomial bloodstream infections. Its resistance to many routinely used antimycotics and its close relationship to the model organism Saccharomyces cerevisiae make this fungus an interesting target of biomedical research. Although the genome sequence was published a decade ago, little is known about the transcriptional dynamics within this pathogen, especially compared to other pathogenic and nonpathogenic yeasts.We provide a detailed RNA-Seq-based analysis of the transcriptomic landscape of C. glabrata in nutrient-rich media as well as under stress conditions and weak acidic and alkaline environments. Using state of the art bioinformatics tools, we were able to refine the annotation of the C. glabrata genome up to 5288 transcriptional active protein-coding ORFs. Introns were identified in 175 of these genes. Based on our data we were able to increase the number of noncoding RNAs to 68. Additionally, we could detect the expression of 50 novel ORFs. This comprehensive analysis of C. glabrata’s transcriptomic landscape significantly enhances the annotation of current genome annotation and contributes to a further understanding of the molecular mechanisms of the pathogenicity of this medical important yeast.

overall_design: Candida glabrata was grown in standard nutrient-rich medium(wt_37), ph4(wt_pH4), ph8(w,_pH8) and in nitrosative stress (GSNO) conditions(wt_GSNO). After harvesting RNA, paired-end and strand-specific RNA-Seq was performed on three biological replicates after PolyA filtering respectively. Additionally, a fourth replicate without filtering was sequenced single-end and not strand-specific..

first: Seána

last: Dugga

first: Ronny

last: Martin


title: Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma

city: New York

first: Daniela

last: Sia

address: 1425 Madison Avenue

department: Liver Diseases

institute: Icahn school of Medicine at Mount Sinai

type: Genome variation profiling by high throughput sequencing

summary: Intrahepatic cholangiocarcinoma (iCCA) is a fatal bile duct cancer with dismal prognosis and limited therapeutic options. By performing RNA- and exome sequencing analyses we have discovered a novel fusion event, FGFR2-PPHLN1 (16%), and damaging mutations in the ARAF oncogene (11%).

overall_design:
Methods: mRNA and gDNA were exctracted from fresh frozen tumor tissues and corresponding normal tissue (n=8 pairs) from patients with iCCA who underwent surgical resection. RNA-seq was performed using Illumina HiSeq 2500 System with 100 nucleotide single-end reads. One sample and its paired non-tumoral tissue were eliminated from the subsequent analysis because of bad RNa quality. The same 8 paired tumors were also analyzed by whole-exome seq.
*** Submitter confirms there are no patient privacy concerns with these data. ***

first: Bojan

last: Losic


title: Epigenetic modifications and chromosome conformations of the beta globin locus throughout development

city: Seattle

first: Kai-Hsin

last: Chang

state: WA

address: 1705 NE Pacific St.

institute: University of Washington

summary: Human embryonic stem cells provide an alternative to using human embryos for studying developmentally regulated gene expression. The co-expression of high levels of embryonic epsilon and fetal gamma globin by the hESC-derived erythroblasts allows the interrogation of epsilon globin regulation at the transcriptional and epigenetic level which could only be attained previously by studying cell lines or transgenic mice. In this study, we compared the histone modifications across the beta globin locus of the undifferentiated hESCs and hESC-, FL-, and mobilized PB CD34+ cells-derived erythroblasts, which have distinct globin expression patterns corresponding to their developmental stages. We demonstrated that the histone codes employed by the beta globin locus are conserved throughout development. Furthermore, in spite of the close proximity of the epsilon globin promoter, as compared to the gamma or beta globin promoter, with the LCR, a chromatin loop was also formed between the LCR and the active epsilon globin promoter, similar to the loop that forms between the gamma or beta globin promoters and the LCR, in contrary to the previously proposed tracking mechanism.

overall_design: Human embryonic stem cells, hESC-, FL-, and PB derived erythroblasts were studied. The enrichment of H3K4me3 and AcH3 acrossed the beta globin locus was studied using ChIP-seq.

first: Xiangdong

last: Fang

first: Hao

last: Wang

first: Andy

last: Huang

first: Hua

last: Cao

first: Yadong

last: Yang

first: Halvard

last: Bonig

first: John

last: Stamatoyannopoulos

first: Thalia

last: Papayannopoulou


title: Pregnancy-associated alterations in DNA methylation patterns of mammary epithelial stem cells [mRRBS]

city: Cambridge

first: Kendell

last: Clement

state: MA

address: 7 Divinity Ave.

institute: Harvard University

summary: Mammary gland development and luminal differentiation occur largely postnatally during puberty and pregnancy. We found that pregnancy had the most significant effects on stem cells, inducing a distinct epigenetic state that remained stable through life.

overall_design: Mammary glands were collected from mice at non-pregnant and pregnant stages for DNA extraction and DNA methylation analysis via mRRBS (multiplexed reduced representation bisulfite sequencing).

first: Sung

last: Huh

first: Patrick

last: Boyle

first: Alexander

last: Meissner

first: Kornelia

last: Polyak


title: Pregnancy-associated alterations in DNA methylation patterns of mammary epithelial stem cells

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


title: CDK7-dependent Transcriptional Addition in Basal-like Breast Cancer (ChIP-Seq)

first: Richard

last: Young

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: Basal-like breast cancer (BBC) is a highly aggressive form of breast cancer that exhibits extremely high levels of genetic complexity and yet a relatively uniform transcriptional program. We postulate that BBC might be highly dependent on uninterrupted transcription of a key set of genes within this gene expression program and might therefore be exceptionally sensitive to inhibitors of transcription. Utilizing a novel kinase inhibitor and CRISPR/Cas9-mediated gene editing, we show here that basal but not luminal breast cancer cells are exceptionally dependent on CDK7, a transcriptional cyclin-dependent kinase. BBC cells are unique in their dependence on this transcriptional CDK and suffer apoptotic cell death upon CDK7 inhibition. An “Achilles cluster” of BBC-specific genes are extremely sensitive to CDK7 inhibition and frequently associated with super-enhancers. We conclude that CDK7 mediates transcriptional addiction to a vital cluster of genes in BBC and CDK7 inhibition may be useful therapy for this challenging cancer.

overall_design: ChIP-Seq for H3K27ac in basal-like breast cancer and luminal-like breast cancer cell lines

first: Yubao

first: Nicholas

last: Kwiatkowski

first: Brian

last: Abraham

first: Tong

middle: I

last: Lee

first: Jean

last: Zhao

first: Nathanael

last: Gray


title: In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs

status: Public on Jan 21 2015

city: Heidelberg

first: Alexis

last: Maizel

address: Im Neuenheimer Feld 230

department: Centre for Organismal Studies

institute: University of Heidelberg

summary:
Background: Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5' cap structure and their subsequent 5’ to 3’ degradation. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA DEPENDENT RNA POLYMERASE 6 (RDR6). PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts.
Results: We show that that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of siRNAs, a subset of which depends on RDR6 for their production.
Conclusions:  Our results suggest that the decapping of aberrant endogenous RNA in P-bodies limits their entry into the PTGS pathway and prevents the subsequent deleterious consequences arising from this entry. We anticipate that the siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs that we coin rqc-siRNAs because they accumulate when RQC processes are impaired.

overall_design: Small RNA-seq experiments performed in duplicates for each condition.

first: Hervé

last: Vaucheret


title: The Ino80 complex prevents invasion of euchromatin into silent chromatin

city: Los Angeles

first: Siavash

middle: K

last: Kurdistani

state: CA

address: 615 Charles E Young Dr South

department: Biological Chemistry

institute: UCLA

summary:
Purpose: We want to know whether Ino80C contribute to chromatin silencing at both euchromatin and heterochromatin
Methods: All yeast cells were collected in exponential phase. Total RNA was extracted with phenol, digested with DNase and further purified by Trizol. Libraries of mRNA were prepared with Illumina TruSeq RNA Sample Prep Kits v2 or stranded RNA Sample Prep Kits. Libraries were sequenced on Illumina HiSeq 2000. ChIP DNA were purified through chromatin immunoprecipitation using an Arp5, H3K79me3 and H3K4me3 antibody.  Libraries were prepared with a KAPA LTP kit and sequenced using the Illumina HiSeq 2000 platform.
Conclusion: Our ChIP-Seq and  mRNA-Seq data show that Ino80C contributes to silent chromatin in both euchromatin an heterochromatin

overall_design: mRNA profiles of  WT_CY337 and ino80Δ, WT_BY4741, sir3Δ, arp5Δ, ies6Δ, ies2Δ sir3Δ-arp5Δwere generated by deep sequencing, using Illumina HiSeq 2000. WT_BY4741, sir3Δ, arp5Δ, ies6Δ, ies2Δ were duplicates.  Enrichment of Arp5, H3K79me3 and H3K4Me3 on chromatin were generated by ChIP-Seq

first: Yong

last: Xue

first: Michael

last: Carey

first: Craig

last: Peterson


title: A transcriptomic analysis of Neurospora crassa using five major crop residues and the novel role of the sporulation regulator rca-1 in lignocellulase production

city: Tianjin

first: Bang

last: Wang

address: 32 Xi Qi Dao, Tianjin Airport Economic Area

country: China

institute: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

summary: Comparative transcriptional profiling of N. crassa grown on five major crop straws of China (barley, corn, rice, soybean and wheat straws) revealed a highly overlapping group of 430 genes, the Biomass commonly Induced Core Set (BICS). A large proportion of induced carbohydrate-active-enzyme (CAZy) genes (82 out of 113) were also conserved across the five plant straws. Excluding 178 genes within the BICS that were also up-regulated under no-carbon conditions, the remaining 252 genes were defined as the Biomass Regulon (BR). Interestingly, 88 genes were only induced by plant biomass and not by three individual polysaccharides (Avicel, xylan, and pectin); these were denoted as the Biomass Unique Set (BUS). Deletion of one BUS gene, the transcriptional regulator rca-1, significantly improved lignocellulase production using plant biomass as the sole carbon source, possibly functioning via de-repression of the regulator clr-2. Thus, this result suggests that rca-1 is a potential engineering target for biorefineries, especially for plant biomass direct microbial conversion processes.

overall_design: Conidia of Neurospora crass wild type were inoculated at 10^6 conidia/mL into 100 mL 1×Vogel’s salts with 2% (w/w) ground crop straws, barley straw, corn straw, rice straw, soybean straw and wheat straw respectively for 30 h or 2% sucrose for 16 h. Then, mycelia were harvested through filtration and immediately frozen in liquid nitrogen.Total RNA from frozen sample was isolated with TRIzol reagent (Invitrogen) and further treated with DNase I (RNeasy Mini Kit, QIAGEN). The qualified RNA was prepared with standard protocol from Shenzhen BGI (China) and sequenced on the Illumina HiSeqTM 2000 platform.

first: Chaoguang

last: Tian


title: Transcriptome Analysis of Populus x canadensis in Response to Drought Stress

city: Pisa

first: Elena

last: Barghini

state: PI

address: Via del Borghetto 80

department: Department of Agriculture, Food and Environment

country: Italy

institute: University of Pisa

summary: To investigate the response of poplar hybrids to drought, leaves were collected from plants to which water was suspended for 8 and 13 days. After measuring the respective relative water content, RNAs were isolated from leaves of moderately and severely droughted plants and from control plants, and Illumina RNA sequencing was performed to analyze RNA synthesis in these tissues.

overall_design: Transcriptional profiling of leaves of poplar plants subjected to 8 or 13 days of water deprivation. Sequence data were generated by deep sequencing, on four replicates (2 per 2 hybrids), using Illumina HiSeq2000.

first: Rosa

last: Cossu

first: Andrea

last: Cavallini

first: Tommaso

last: Giordani


title: Deep sequencing of the murine olfactory receptor neuron transcriptome

city: Bochum

first: Ninthujah

last: Kanageswaran

address: Universitätsstr. 150

department: Biology and Biotechnology

institute: Ruhr-University Bochum

summary: The recent development of next-generation sequencing (NGS) techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS)-sorted olfactory receptor neurons (ORNs) obtained from transgenic OMP-GFP mice

overall_design: The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome.

first: Benjamin

last: Schreiner

first: Sabrina

last: Baumgart

first: Janine

last: Altmüller

first: Christian

last: Becker

first: Hanns

last: Hatt

first: Günter

last: Gisselmann


title: Deconvolving the recognition of DNA shape from sequence

first: Harmen

last: Bussemaker

state: NY

address: 1212 Amsterdam Ave MC 2441

department: Biological Sciences

institute: Columbia University

summary: Binding of transcription factors to DNA is mediated by the recognition of the chemical signatures of the DNA bases and the three-dimensional shape of the DNA molecule.  The direct contribution of DNA shape to DNA-binding specificity has been difficult to assess, as DNA shape is a consequence of its sequence.  Here, we teased apart these two modes of recognition in the context of Hox-DNA binding.  We made a series of mutations in Hox residues that, in a co-crystal structure, only recognize DNA shape, and tested the effect on DNA binding preferences using SELEX-seq.  Analysis of shape features of selected sequences revealed that these residues are both necessary and sufficient for selection of sequences with distinct shape features.   We used statistical machine learning to show that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence.  We conclude that shape readout is a direct and critical component of binding site selection by Hox proteins.

overall_design: Three rounds of SELEX were performed on a series of Hox mutants as described in Slattery et al, Cell, 2011 (PMID 22153072) and Riley et al, Methods in molecular Biology, 2014 (PMID 25151169).  Briefly, His-tagged Scr and Antp mutant proteins were incubated with a randomized 16mer oligonucleotide library, and bound DNA was amplified and sequenced as described (PMID 22153072, PMID 25151169).

first: Richard

last: Mann

first: Remo

last: Rohs

first: Matthew

last: Slattery


title: Ligand-Dependent Enhancer Activation Regulated by Topoisomerase-I Activity

city: La Jolla

first: Michael

middle: G

last: Rosenfeld

address: 9500 Gilman Drive

department: School of Medicine

institute: University of California, San Diego

summary: The discovery that enhancers are regulated transcription units, encoding eRNAs, has raised new questions about the mechanisms of their activation. Here, we report an unexpected molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis. Using dihydrotestosterone (DHT)-induced binding of androgen receptor (AR) to prostate cancer cell enhancers as a model, we show rapid recruitment, within minutes, of DNA topoisomerase I (TOP1) to a large cohort of AR-regulated enhancers. Furthermore, we show that the DNA nicking activity of TOP1 is a prerequisite for robust eRNA synthesis and enhancer activation and is kinetically accompanied by the recruitment of ATR and the MRN complex, followed by additional components of DNA damage repair machinery to the AR-regulated enhancers. Together, our studies reveal a linkage between eRNA synthesis and ligand-dependent TOP1-mediated nicking - a strategy exerting quantitative effects on eRNA expression in regulating AR-bound enhancer-dependent transcriptional programs.

overall_design:
Genome-wide binding analysis of AR, TOP1, MRE11 in prostate cancer cell line LNCaP with or without 5alpha-dihydrotestosterone (DHT) treatment.
Nascent RNA analysis by global nuclear run-on (GRO-seq) in LNCaP cells transfected with siRNA with or without DHT treatment.
Distribution of transcriptionally engaged  RNA Pol II in LNCaP cells with or without DHT treatment by precision nuclear run-on and sequencing (PRO-seq).

first: Janusz

last: Puc

first: Piotr

last: Kozbial

first: Wenbo

last: Li

first: Yuliang

last: Tan

first: Zhijie

last: Liu

first: Tom

last: Suter

first: Kenneth

last: Ohgi

first: Jie

last: Zhang

first: Aneel

last: Aggarwal


title: Identification of early response genes to Pratylenchus coffeae in resistant ramie by digital gene expression analysis

status: Public on Jan 20 2015

city: Changsha

first: Yongting

last: Yu

address: No 348, Xianjiahu West Rd.

institute: Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science

summary: Digital gene expression (DGE) analysis was performed to study differentially expressed genes (DEGs) between CK and P. coffeae challenged (CH) roots of ramie. A total of 10.16 and 8.07 million clean reads were detected in the CK and CH libraries, respectively. A total of 137 genes were identified as DEGs, 117 and 20 of them were up- and down- regulated respectively, in response to P. coffeae infection.

overall_design: Roots samples of Pratylenchus coffeae infected(E2) and un-infected (E1) ramie were RNA-Seq sequenced to compare differented expressed genes.

first: Aiguo

last: Zhu


title: Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation [ChIP-Seq]

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

summary: DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined chromosomal binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects chromosomal binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.

overall_design: Genome-wide binding analysis for biotin-tagged DNMT3A2 and DNMT3B and variants in wild type ES, wild type neuroprogenitor cells, ES cells triple-KO for Dnmt1,3a,3b and ES cell mutant for Setd2

first: Tuncay

last: Baubec

last: Schübeler


title: Expression data from the Arabidopsis with freezing treatment following the inhibitors of DNA methylation and histone deacetylation

city: Lanzhou

first: Yuan

last: Song

state: Gansu

address: the South of Tianshui Road

department: the school of life science

institute: Lanzhou University

summary:
To understand the roles of DNA methylation and histone deacetylation in plant gene network that controls plant tolerance to freezing treatment, we used Affymetrix GeneChips ATH-121501 to analysis.
For microarray analysis, Arabidopsis (ecotype Columbia) seedlings grew in four types of sterile growth medium: no any other modifying agents, added 7g/ml 0.5 M aza-dC in water (DNA methylation inhibitor), added TSA in methanol (histone deacetylation inhibitor), both aza-dC and TSA, all for 16 day in growth chamber (16-h day length at 70% relative humidity, 23 °C) , then all seedlings were cold treated at 0 °C for 24 h in growth chamber (16-h day length at 70% relative humidity). A total of 3305 genes expression were statistically analysized. Our study provides some clues that the transcript levels of some cold-responsive genes regulated by DNA methylation and histone deacetylation. This will be valuable for understanding gene regulation by epigenetic modifications under freezing stress.  Keywords: Cold Stress response, DNA methylation, histone deacetylation

overall_design: Three replicates for aza-dc and TSA treatment, and two replicates for Mock and both aza-dc and TSA treatment. All samples were treated 0°C 24h.

first: Lining

last: Tian


title: Defining CD4 T Cell Memory by the Epigenetic Landscape of CpG DNA Methylation [Bisulfite-Seq]

first: Daniel

last: Salomon

address: 10550 N Torrey Pines Rd, MEM-241

department: Molecular & Experimental Medicine

institute: The Scripps Research Institute

summary: Memory T cells are primed for rapid responses to antigen; however, the molecular mechanisms responsible for priming remain incompletely defined. CpG methylation in promoters is an epigenetic modification, which regulates gene transcription. Using targeted bisulfite sequencing, we examined methylation of 2100 genes (56,000 CpG) mapped by deep sequencing to T cell activation in human naïve and memory CD4 T cells. 466 CpGs (132 genes) displayed differential methylation between naïve and memory cells. 21 genes exhibited both differential methylation and gene expression before activation, linking promoter DNA methylation states to gene regulation; 6 genes encode proteins closely studied in T cells while 15 genes represent novel targets for further study. 39 genes exhibited reduced methylation in memory cells coupled with increased gene expression with activation compared to naïve cells, revealing specific genes more rapidly expressed in memory compared to naïve cells and potentially regulated by DNA methylation. These findings define a DNA methylation signature unique to memory CD4 T cells and correlated with activation-induced gene expression.

overall_design: Targeted bisulfite sequencing of primary human naïve and memory CD4 T cells at rest and 48 hours post-activation.

first: H

last: Komori

first: G

last: Hart

first: Sarah

last: LaMere

first: Pamela

middle: V

last: Chew


title: Genome-wide maps of the effect of antimicrobial compounds in P. putida DOT-T1E

city: Malaga

first: Carlos

middle: Molina

last: Santiago

state: Malaga

address: Bulevar Louis Pasteur 49

department: MIcrobiology

country: Spain

institute: UMA

summary: We report the application of a high-throughput technique, RNA-seq, to study the transcriptomic response of P. putida DOT-T1E in the presence of antibiotics with different mechanisms of action with the aim to study in more detail the defense mechanisms that bacteria use to resist against toxic compounds. We find that P. putida DOT-T1E responde in a different way against each antimicrobial compound, what clearly shows that bacteria defense in different ways depending on the targets that compounds uses to attack. Our work is the first global transcriptomic analysis done in P. putida DOT-T1E in the presence of a considerable range of antibiotics.

overall_design: P. putida DOT-T1E mRNA profiles in the presence of control condition (LB) and 8 different antibiotics (ampicillin, chloramphenicol, kanamycin, ciprofloxacin, tetracycline, spectinomycin, gentamicin and rifampicin)

last: Molina-Santiago

first: Juan

middle: L

last: Ramos


title: Defining CD4 T Cell Memory by the Epigenetic Landscape of CpG DNA Methylation [RNA-Seq]

overall_design: RNA sequencing of primary human naïve and memory CD4 T cells at rest and 48 hours post-activation.

middle: T

middle: A


title: Defining CD4 T Cell Memory by the Epigenetic Landscape of CpG DNA Methylation

overall_design: Refer to individual Series


title: Transcriptome profile of oocytes expressing a catalytically inactive knock-in allele of Ago2

city: Research Triangle Park

first: Paula

last: Stein

address: 111 T.W. A;exander Dr.

department: Reproductive and Developmental Biology Laboratory

institute: NIEHS

summary: The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because miRNA function is suppressed in mouse oocytes, it has been proposed that endo-siRNAs may have a role during female meiosis. By utilizing a catalytically inactive knock-in allele of AGO2 specifically in oocytes, we disrupt the function of siRNAs and analyze the transcriptome of these oocytes in comparison with Ago2 null, and Dicer null oocytes.

overall_design:
Germinal vesicle-intact, full-grown oocytes were collected from eCG-primed females expressing a catalytically inactive knock-in allele of Ago2 (Ago2 ADH) specifically in oocytes. Oocytes were also collected from Ago2 null and Ago2 fl/fl oocytes, as well as Dicer wild-type and null mice. Oocytes were freed of attached cumulus cells by pipetting. Twenty oocytes collected from 3 different mice were used for high-throughput RNA sequencing. Three replicates for all genotypes except Ago2 null (two).
The mouse background strain is "mostly" C57BL6, i.e., it is a cross between 3 different transgenic mice of different strains.

first: Nikolay

last: Rozhkov

first: Fan

last: Li

first: Brian

last: Gregory

first: Gregory

middle: J

last: Hannon

first: Richard

last: Schultz


title: Base-resolution maps of 5-formylcytosine and 5-carboxylcytosine reveal genome-wide DNA demethylation dynamics

city: Chicago

first: Dali

last: Han

state: IL

address: 5801 South Ellis Avenue

department: Department of Chemisty

institute: University of Chicago

summary: 5-methylcytosine (5mC), the predominant epigenetic modification on DNA, plays critical roles in mammalian development and is dysregulated in various human pathologies. In mammals, the TET family of dioxygenases can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in a stepwise manner. 5fC and 5caC are selectively recognized and excised by mammalian thymine DNA glycosylase (TDG), and restored to normal cytosine through base excision repair (BER). Once 5mC/5hmC is converted to 5fC and/or 5caC, the modified cytosine is committed to demethylation through BER. Thus 5fC and 5caC most likely mark active demethylation in the mammalian genome. Here we introduce a genome-wide approach to obtain single-base resolution maps of 5fC and 5caC, respectively. We show that, in mouse embryonic stem cells (mESCs), 5fC and 5caC are preferentially generated at highly hypomethylated regions and more active enhancers. Moreover, 5caC-marked regions are characterized by the lowest methylation and highest enhancer activity among all modification sites associated with 5hmC, 5fC and 5caC, and are enriched adjacent to pluripotency transcription factor (TF)-binding motifs. These observations, together with the surprising lack of overlap between 5fC and 5caC sites, highlight a gradient of Tet-mediated 5mC oxidation activity at regulatory elements in tuning epigenetic dynamics11.

overall_design: DNA immunoprecipitation coupled chemical-modification assisted bisulfite sequencing (DIP-CAB-Seq) for Tdg fl/fl and Tdg-/- mESCs

first: Xingyu

last: Lu

first: Boxuan

last: Zhao

first: Chuan

last: He


title: Transcriptome analyses of dBRWD3 mutant, and dBRWD3, yem double mutant brain

status: Public on Jan 19 2015

city: Taipei

first: June-Tai

last: Wu

state: (Click to Select US State)

address: No.4, Sec. 1, Roosevelt Rd.

country: Taiwan

institute: National Taiwan Univeristy

summary: We report the high-throughput profiling of brain RNA from three Drosophila stains: dBRWD3PX2/+, dBRWD3PX2/PX2 and dBRWD3PX2/PX2, yemGS21861/GS21861. By obtaining over 50 million reads of sequence, WE compared the transcriptomic differences among the brains from these three stains. We found that the expression of 871 genes was significantly different between heterozygous control and homozygous dBRWD3 mutant brains (484 upregulated genes, 387 downregulated genes, p<0.05). Gene ontology (GO) analysis of the 871 genes revealed a broad spectrum of biological processes, ranging from synaptic activity to housekeeping metabolism subjective to dBRWD3 regulation.  Among the 387 downregulated genes, the expression of 360 genes (92.8%) was increased in the dBRWD3, yem double mutant brains compared with dBRWD3 mutant. Among the 484 upregulated genes, the expression of 412 genes (85.1%) was decreased in the double mutant brains. These differential genes were evenly distributed on X chromosome and autosomes (149 on X, 178 on 2L, 154 on 2R, 166 on 3L, and 207 on 3R). These analyses indicate that dBRWD3 regulates gene expression in the brain mainly through the HIRA/YEM complex.

overall_design: Examination of brain transcriptome in 3 Drosophila strains.

first: Chien-Yu

last: Chen


title: Stress-induced endogenous siRNAs targeting regulatory intron sequences in Brachypodium

city: Atlanta

first: Hsiao-Lin

middle: V.

last: Wang

state: GA

address: 615 Michael Street,, Whitehead Biomedical Research Building, Room 355,

department: School of Medicine, Department of Human Genetics

institute: Emory University

summary:
ABSTRACT: Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional 70 and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects
on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited.
Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon
plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group 75 of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3′
UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions
and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr- siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements 80 through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially
in other eukaryotes.

overall_design: Analysis of smRNA populations in Brachypodium plants challenged by abiotic stresses: To profile the populations of smRNAs in the model monocot Brachypodium distachyon and examine their regulation in response to abiotic stresses, we conducted high-throughput sequencing of small RNAs from plants exposed to four different abiotic stress conditions, cold, heat (air), heat (water immersion), and salt, in the wild type Brachypodium cultivar Bd21. For our experiments we used information from the literature to select two time-points for stress durations, short and long, which differed for each stress: cold (6 and 24 hours), heat-air (1 and 3 hours), heat-water (1 and 3 hours), and salt (48 hours). We generated small RNA libraries for Illumina sequencing (GAII) from the leaves of Brachypodium plants subjected to stresses and selected smRNAs between 15 and 40 nt in length, which we mapped to the Brachypodium genome.

first: Julia

last: Chekanova


title: Knock-in of PIK3CA-H1047R into MCF-10A

first: Jonathan

middle: Ross

last: Hart

address: 10550 North Torrey Pines Road, BCC239

department: Molecular and Experimental Medicine

summary: We have compared the proteome, transcriptome and metabolome of two isogenic cell lines: MCF-10A, derived from human breast epithelium, and the mutant MCF-10A-H1047R. These cell lines differ by a single amino acid substitution (H1047R) caused by single nucleotide change in one allele of the PIK3CA gene which encodes the catalytic subunit p110α of phosphatidylinositol 3-kinase (PI3K). The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse; with examples in structural protein levels, the DNA repair machinery and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.

overall_design: 2 cell lines (H1047R and WT), 4 time points (0, 6, 12, 24 hours), 3 replicates


title: Investigation of the role of histone modification propagating activity of GLP

city: Beijing

first: Zhuqiang

last: Zhang

address: 15 Datun Road, Chaoyang District.

institute: Institute of Biophysics, CAS

summary:
We use ChIP-Seq and RNA-Seq technology to profile the H3K9me2 modification and transcription under different conditions of GLP activity.
GLP and G9a are major H3K9 dimethylases, and are essential for mouse early embryonic development. Here we report that GLP and G9a possess intrinsic histone methylation propagating activities. The histone methyltransferase activities of GLP and G9a are stimulated by neighboring nucleosomes pre-methylated at H3K9. These stimulation events function in cis and are dependent on H3K9 methylation binding activities of ankyrin repeats domains in GLP and G9a. In mouse embryonic stem cells (ESCs) harboring a mutant GLP lacking H3K9 methylation propagating activity, pluripotent genes display a delayed kinetics in establishing H3K9 methylation and gene silencing during differentiation. Disruption of the H3K9 methylation propagating activity of GLP in mice causes growth retardation of the embryos, ossification defects of calvaria and early postnatal lethality. We propose that GLP¡¯s ability to rapidly propagate H3K9 methylation is required for efficient gene silencing during programmed cell fate transition.

overall_design: H3K9me2 and H3K9me1 are ChIPped and sequenced in WT mESC and GLP-mutant mESCs, and RNA-Seq was done for those cells as well. ChIP-seq for H3K9me2 was also performed in wildtype and mutant newborn mouse liver.

last: Liu

first: Hui

first: Yonghua

last: Jiang

first: Jun

last: Xiong

first: Zuodong

first: Xiaohua

last: Zhou

first: Jia

first: Hong

first: She

first: Tao

last: Cai

first: Shaorong

last: Gao

first: Bing

last: Zhu


title: Transcriptome analysis of 5 red Italian grapevine varieties during berry development

city: Verona

first: Mélanie

last: Massonnet

state: Verona

address: Strada Le Grazie, 15

department: Biotechnology Department

institute: University of Verona

summary: We report the berry pericarp transcriptomic profiles of 5 red Vitis vinifera varieties: Sangiovese, Barbera, Negro amaro, Refosco and Primitivo at 4 growth stages: pea-sized berries (Bbch 75) at almost 20 days after flowering, berries beginning to touch (Bbch77) just prior véraison, the softening of the berries (Bbch 85) at the end of véraison and berries ripe for harvest (Bbch 89)

overall_design: mRNA profiles of 5 red grapevine varieties at 4 growth stages were generated by deep sequencing, in triplicate, using Illumina Hiseq 1000.

first: Mario

last: Pezzotti


title: The Integrator complex controls the termination of transcription at diverse classes of gene targets

city: Frederick

first: Xiaolin

state: MD

address: 8560 Progress Drive, C3001

department: Cancer Research Technology Program

institute: Frederick National Laboratory for Cancer Research/SAIC-Frederick Inc

summary: Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown.  Using affinity purifications and HIV Integration Targeting-Sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes.  Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination.  However, HIT-Seq reveals that Integrator also binds to the 3’ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts.  Depletion of Integrator subunits results in transcription termination failure, disrupting histone mRNA processing, and leading to polyadenylation of snRNAs and histone mRNAs.   Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated.  Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites.

overall_design: HITseq was used to interrogate chromatin binding sites of of proteins in the complex (including INIP, INTS3, INTS9, INTS11, NABP1, NABP2, NELFA, NELFB, NELFCD, NELFE, SPT5).   These proteins were fused to LEDGF-IBD and expressed in mouse LEDGF-/-MEF cells.  The fusion proteins then target HIV integration to the chromatin binding sites.  The HIV integration sites were identified using linker-mediated PCR and highthroughput sequencing and serve as surrogate for chromatin binding sites. Mapping was done using mouse genome mm9.

first: Jeffrey

middle: R

last: Skaar

first: Andrea

last: Ferris

first: Anita

last: Saraf

first: Kum

last: Khanna

first: Laurence

last: Florens

first: Michael

middle: P

last: Washburn

first: Stephen

middle: H

last: Hughes

first: Michele

last: Pagano


title: Transcriptome analysis of luminal and basal cell subpopulations in the lactating versus pregnant mammary gland

city: Parkville

first: Gordon

last: Smyth

state: Vic

address: 1G Royal Pde

department: Bioinformatics

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

summary: To identify genes specifically expressed in lactating mammary glands, the gene expression profiles of luminal and basal cells from different developmental stages were compared.

overall_design: Comparison of gene expression in luminal and basal cells harvested from the mammary glands of virgin, 18.5 day pregnant and 2 day lactating mice (2 mice per stage).

first: Nai

middle: Y

last: Fu

first: Aaron

last: Lun

first: Jane

middle: E

last: Visvader


title: FASCIATED EAR4 encodes a bZIP transcription factor that controls shoot meristem size in maize

city: St. Louis

last: Eveland

state: MO

address: 975 N. Warson Road

institute: Donald Danforth Plant Science Center

summary: fasciated ear4 (fea4) is a semi-dwarfed mutant with fasciated ears and tassels, and greatly enlarged vegetative and inflorescence meristems.  Chromatin Immunoprecipitation-sequencing (ChIP-seq) and expression profiling by RNA-seq suggest that fea4 is required to regulate the auxin response and leaf differentiation programs in the periphery of the meristem, suggesting a new mechanism of meristem size regulation that is spatially and mechanistically distinct from the CLV-WUS model.

overall_design: To gain insight into transcriptional regulation by FEA4, we used RNA-seq to profile genome-wide expression changes in developing ear primordia of fea4 loss-of-function mutants compared to fea4/+ wild-type (wt) siblings.  To map genome-wide occupancy of FEA4 and define its putative transcriptional targets, we performed ChIP-seq using the pFEA4-YFP::FEA4 transgenic lines.

last: Pautler

first: David

last: Jackson


title: Transposon defense by endo-siRNAs, piRNAs and somatic pilRNAs: Roles of Loqs-PD and R2D2

city: Munich

first: Klaus

last: Förstemann

address: Feodor-Lynen 25

department: Gene Center

institute: Ludwig-Maximilians University

summary: Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing mechanisms that repress TEs expression: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2 that primarily helps to direct siRNAs for loading into Ago2. We provide deep sequencing evidence consistent with the idea that R2D2 and Loqs-PD can function in part redundantly. Certain transposons display a preference for either dsRBD-protein for production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in the germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway.

overall_design: small RNA sampling experiment; small RNAs were prepared from head & thorax as well as dissected ovaries of Adult female Drosophila melanogaster. We used homozygous mutants of the dsRBD proteins Loqs and r2d2 to determine their contribution to the biogenesis of transposon-derived small RNAs. Heterozygous mutant animals served as control. For each RNA sample, we performed one deep-sequencing run without any treatment, and in parallel one sequencing run after periodate oxidation and beta-elimination. After this treatment, only Ago2, Piwi, Aub and Ago3-loaded small RNAs remain as they carry a 2'-O-methyl modification at their 3'-end. This helps to determine the loading status of the small RNAs detected. In total 8 different RNA samples were prepared and 16 libraries were sequenced.

first: Milijana

last: Mirkovic-Hösle


title: Cytosine methylation data generated by HELP-tagging from human osteosarcoma biopsy samples

status: Public on Jan 18 2015

city: Valhall

first: Jeremy

last: Rosenblum

address: 40 Sunshine Cottage Rd, Skyline Office 1N-H13

institute: New York Medical College

summary: Osteosarcoma is the most common primary malignant bone tumor in children.  Validated markers for disease prognosis available at diagnosis are lacking.  No genome-wide DNA methylation studies linked to clinical outcomes have been reported in osteosarcoma.  To address this, we tested the methylome at over 1.1 million loci in 15 osteosarcoma biopsy samples obtained prior to the initiation of therapy and correlated these molecular data with disease outcomes.  At the tested loci, samples obtained from patients who experienced disease relapse were generally more methylated than those from patients who did not have recurrence.  In samples from patients who went on to have recurrent disease, increased DNA methylation was found at gene bodies, intergenic regions and empirically-annotated candidate enhancers, whereas candidate gene promoters were unusual for a more balanced distribution of increased and decreased DNA methylation.  A locus at the TLR4 gene demonstrates one of strongest associations between DNA methylation and five year event-free survival, with empirical annotation of this locus showing promoter characteristics.  Our data indicate that DNA methylation information has potential to be predictive of outcome in pediatric osteosarcoma, and that both promoters and non-promoter loci are potentially informative in DNA methylation studies.

overall_design: 15 samples.  HpaII libraries were compared to at least 3 MspI libraries from the same sample

first: N

last: Wijetunga

first: Melissa

last: Fazzari

first: Donald

last: Barskauskas

first: Mark

last: Krailo

first: Richard

last: Gorlick

first: John

last: Greally


title: Transcriptome analysis of Oshsfa2e mutant plants under controlled drought stress and well-watered conditions at vegetative stage

status: Public on Jan 16 2015

city: Fayetteville

first: Andy

last: Pereira

state: AR

address: 115 Plant Science Building,

department: Crop, Soil and Environmental Sciences (CSES)

institute: University of Arkansas,

summary: Heat shock factors (Hsfs) are known to regulate heat and drought stress response by controlling the expression of heat shock proteins and oxidative stress responsive genes. Loss-of-function of OsHSFA2e gene resulted in increased sensitivity of rice plants to drought and heat stress. To identify the targets of OsHSFA2e and dissect the stress response pathway regulated by it, we performed transcriptome profiling of Oshsfa2e mutant plants under drought stress as well as well-watered conditions by RNA-sequencing.

overall_design: OsHSFA2e loss-of-function rice plants (Oryza sativa ssp. japonica cv. Nipponbare) were exposed to controlled drought stress and well-watered conditions at the vegetative stage. Controlled drought (DR) stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Another set of plants were maintained at 100% FC as well-watered (WW) condition. Total RNA isolated from leaf tissue was used for RNA-sequencing. Two biological replicates per sample were sequenced. cDNA library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Sequencing was carried out on each library to generate 50 bp SE reads using Illumina High-Seq 2000 platform. The transcriptome reads were mapped to the rice reference genome sequence (MSU 7.0) with tophat1.3.1 using the program’s default parameters (http://tophat.cbcb.umd.edu). Mapped RNA-Seq reads were assembled into transcripts by Cufflinks (http://cufflinks.cbcb.umd.edu/) and differentially expressed genes were identified by using Cuffdiff.

first: Ramegowda

last: Venkategowda

first: Subodh

last: Srivastava


title: Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation [Bisulfite-Seq]

city: Basel

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

institute: Friedrich Miescher Institute for Biomedical Research

summary: DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined chromosomal binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects chromosomal binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.

overall_design: Whole-genome bisulfite sequencing for Dnmt1,3a,3b-triple-KO ES cells expressing DNMT3A2 or DNMT3B1 and for Dnmt1,3a,3b,Setd2-KO ES cells expressing DNMT3B1

first: Tuncay

last: Baubec

last: Schübeler


title: Drosophila miR-277 controls branched-chain amino acid catabolism and affects lifespan

summary: Development, growth and adult survival are coordinated with available metabolic resources. The insulin/IGF and TOR signaling pathways relay nutritional status, thereby ascertaining that the organism responds appropriately to environmental conditions. MicroRNAs are short (21-23 nt) regulatory RNAs that confer specificity on the RNA-induced silencing complex (RISC) to inhibit a given set of mRNA targets. We profiled changes in miRNA expression during adult life in Drosophila melanogaster and determined that miR-277 is down-regulated with age. This miRNA controls branched-chain amino acid (BCAA) catabolism and the activity of the TOR kinase, a central growth regulator. Metabolite analysis suggests that the mechanistic basis may be an accumulation of BCKAs, rather than BCAAs, thus avoiding potentially detrimental consequences of increased branched chain amino acid levels on e.g. translational fidelity. Constitutive miR-277 expression as well as transgenic inhibition with a miRNA sponge construct shortens lifespan. Furthermore, constitutive miR-277 expression is synthetically lethal with reduced insulin signaling. Thus, optimal metabolic adaptation requires tuning of cellular BCAA catabolism by miR-277 to be concordant with systemic growth signaling.

overall_design: Transgenic Drosophila melanogaster fruitflies carrying strong, ubiquitously expressed pre-miR277 hairpins (wt and two mutant versions) were dissected, total RNA was extracted from the abdomen and gel-purified for size selection (~18-30 nt). Digested plasmid samples were compared to those of circular plasmids and a nontransfected control. The purpose of this experiment was to demonstrate the extent of expression from mutant pre-miR277 hairpins, mut1 should abolish Drosha-processing while mut2 is conservative.


title: Human Promoters Are Intrinsically Directional

city: Berlin-Buch

first: Scott

middle: Allen

last: Lacadie

address: Robert-Rössle-Str. 10

department: Berlin Institute for Medical Systems Biology

institute: Max Delbrück Center for Molecular Medicine

summary: Divergent transcription, in which reverse-oriented transcripts occur upstream of eukaryotic promoters in regions devoid of annotated genes, has been suggested to be a general property of active promoters. Here we show that the human basal RNA polymerase II transcriptional machinery and core promoter are inherently unidirectional, and that reverse-oriented transcripts originate from their own cognate reverse-directed core promoters. In vitro transcription analysis and mapping of nascent transcripts in cells revealed that core promoters are unidirectional and that sequences at reverse start sites are similar to those of their forward counterparts. The use of DNase I accessibility to define proximal promoter borders revealed that about half of promoters are unidirectional and that these unidirectional promoters are depleted at their upstream edges of reverse core promoter sequences and their associated chromatin features. Divergent transcription is thus not an inherent property of the transcription process, but rather the consequence of the presence of both forward- and reverse-directed core promoters.

overall_design: Using 5'-GRO-seq and GRO-seq to determine mechanisms of divergent transcription initiation

first: Sascha

last: Duttke

first: Mahmoud

middle: M

last: Ibrahim

first: Christopher

last: Glass

last: Corcoran

last: Benner

first: Sven

last: Heinz

first: James

last: Kadonaga

first: Uwe

last: Ohler


title: Zinc Finger Protein 407 (ZFP407) Regulates Insulin-Stimulated Glucose Uptake and Glucose Transporter 4 (Glut4) mRNA

city: Cleveland

first: David

last: Buchner

state: OH

address: 10900 Euclid Ave

department: Genetics and Genome Sciences

institute: Case Western Reserve University

summary: Using RNA-Seq, we compared the transcriptomes of differentiated 3T3-L1 adipocytes for control and ZFP407-deficient cells

overall_design: Differentiated 3T3-L1 cells were electroporated with control or 1 of 2 Zfp407 siRNAs. Six independent siRNA electroporations were conducted for the control siRNA and 3 independent electroporations were conducted for each Zfp407 siRNA.

first: Alan

last: Saltiel


title: Genome-wide transcriptome analysis of rice in response to controlled drought stress at vegetative

last: Pereira

summary: In this study, genome-wide transcriptome profiling was used to understand molecular genetic mechanism of drought tolerance in rice. Illumina High-Seq 2000 platform was used for sequencing RNA from leaf tissue of rice plants exposed to controlled drought stress and well-watered conditions. The differentially expressed genes were used to identify biological process and cis-regulatory elements enriched under drought stress compared to well-watered conditions.

overall_design: Oryza sativa ssp. japonica cv. Nipponbare plants were exposed to controlled drought stress and well-watered conditions at the vegetative stage. Controlled drought (DR) stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Another set of plants were maintained at 100% FC as well-watered (WW) condition. Total RNA isolated from leaf tissue was used for RNA-sequencing. Two biological replicates per sample were sequenced. cDNA library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Sequencing was carried out on each library to generate 50 bp SE reads using Illumina High-Seq 2000 platform. The transcriptome reads were mapped to the rice reference genome sequence (MSU 7.0) with tophat1.3.1 using the program’s default parameters (http://tophat.cbcb.umd.edu). Mapped RNA-Seq reads were assembled into transcripts by Cufflinks (http://cufflinks.cbcb.umd.edu/) and differentially expressed genes were identified by using Cuffdiff.

middle: K


title: Deconstruction of DNA methylation patterns during myogenesis reveals specific epigenetic events in the establishment of the skeletal muscle lineage

city: Badalona

first: Miguel

last: Peinado

state: Barcelona

address: Ctra. de Can Ruti, camí de les escoles, s/n

institute: IGTP

summary: Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. We have investigated DNA genome-wide methylation dynamics in embryonic stem cells, primary myoblasts, terminal differentiated myotubes and mature myofibers. About 1.000 differentially methylated regions (DMRs) have been indentified during muscle-lineage determination and terminal differentiation. As a whole, muscle lineage commitment was characterized by a major gain of DNA methylation, while muscle differentiation was accompanied by loss of DNA methylation in CpG-poor regions. Notably, hypomethylated regions in muscle cells were neighboured by enhancer-type chromatin, suggesting the involvement of DNA methylation in the regulation of cell-type specific enhancers. Indeed, one of the hypomethylations detected in muscle cells affected the super-enhancer of the master transcription factor Myf5. Super-enhancers have been defined as large clusters of transcriptional enhancers driving cell-identity and gene expression, but how these lineage-specific super-enhancers are specifically activated or repressed in different tissues is not well understood. We demonstrated that the binding of the transcription factor USF1 to Myf5 locus occurs upon DNA demethylation of the super-enhancer region in myogenic committed cells. Taken all together, we have characterized the unique DNA methylation signatures of muscle-committed cells and highlighted the importance of DNA methylation mediated regulation of cell identity super-enhancers.

overall_design: We have investigated DNA genome-wide methylation dynamics in embryonic stem cells, primary myoblasts, terminal differentiated myotubes and mature myofibers by AIMS-seq techniques and coupled to microarray expression data by SurePrint G3 Mouse 8x60K from Agilent Technologies. Samples were in triplicates, except for  ESCs (quadruplicates).

first: Elvira

last: Carrió

first: Mònica

last: Suelves


title: Comparative transcriptome analysis of Shewanella MR-1 under oxic vs. suboxic conditions

city: West Lafayette

first: Stephen

last: Lindemann

state: IN

address: 745 Agriculture Mall Drive

department: Food Sciences

institute: Purdue University

summary: To understand how cell physiological state affects mRNA translation, we used the bacterium Shewanella oneidensis MR-1 grown under steady state conditions at either 20% or 8.5% O2. Using a combination of quantitative proteomics and RNA-Seq, we generated high-confidence data on >1000 mRNA and protein pairs. By using a steady state model, we found that differences in protein–mRNA ratios were primarily due to differences in the translational efficiency of specific genes. When oxygen levels were lowered, 28% of the proteins showed at least a 2-fold change in expression. Transcription levels were significantly altered for 26% of the protein changes; translational efficiency was significantly altered for 46% and a combination of both was responsible for the remaining 28%. Changes in translational efficiency were significantly correlated with the codon usage pattern of the genes and measurable tRNA pools changed in response to altered O2 levels. Our results suggest that changes in the translational efficiency of proteins, in part due to altered tRNA pools, is a major determinant of regulated alterations in protein expression levels in bacteria. For further details, see the resulting publication and its supplemental material: Taylor, R. C. et al.,  "Changes in translational efficiency is a dominant regulatory mechanism in the environmental response of bacteria", Integrative Biology, 2013, 5, 1393. DOI: 10.1039/c3ib40120k

overall_design: mRNA profiles of Shewanella MR-1 were generated by sequencing on SOLID sequencers under high (20%) or low (8.5%) oxygen, with primary sample comparison pair in triplicate, other samples in duplicate.

first: Ronald

middle: C

last: Taylor

first: Bobbie-Jo

last: Webb-Robertson

first: Lye

last: Markillie

first: Margrethe

last: Serres

first: Bryan

last: Linggi

first: Joshua

last: Aldrich

first: Eric

last: Hill

first: Margaret

middle: F

last: Romine

first: Mary

middle: S

last: Lipton

first: H

last: Wiley


title: Genome-wide sequencing of small RNAs of Echinococcus granulosus shows microRNAs may be involved life cycle stage development and differentiation

city: Shanghai

first: Yun

last: Bai

state: Shanghai

address: Building 10#, No 1278, Baode Road

department: Central Laboratory

institute: Shanghai Skin Disease Hospital

summary: MicroRNAs (miRNAs) are important post-transcriptional regulators which control growth and development in eukaryotic animals. The cestode Echinococcus granulosus has a complex life-cycle involving different development stages but the mechanisms underpinning this development, including the involvement of miRNAs, remain unknown. Using Illumina deep-sequencing technology, we sequenced at the genome-wide level three small RNA populations from the adult, protoscolex and cyst membrane of E. granulosus. A total of 94 miRNA candidates (coding 100 mature miRNAs and 30 miRNA*s) were predicted by a computational pipeline, of which 25 mature miRNAs and 2 miRNA*s were identified experimentally. Through comparison of expression profiles, we found 48 mature miRNAs and 17 miRNA*s expressed in different patterns in the three life stages examined and 31 of them were validated by real-time PCR. Most of the differentially expressed miRNAs exhibited obvious up-regulated expressions in the adult stage and down-regulation in the hydatid cyst. A total of 3,622 genes were predicted to be targets of 126 mature miRNAs and 50 miRNA*s. Further analysis of the differentially expressed miRNAs and their potential targets indicated that they may be involved in bi-directional development, nutrient metabolism and nervous system development in E. granulosus. This study has, for the first time, provided a comprehensive description of the different expression patterns of miRNAs in the three distinct life cycle stages of E. granulosus. Understanding the regulatory processes involving miRNAs in E. granulosus may help in the exploration of the mechanism of interaction between this parasitic worm and its definitive and intermediate hosts and provide new information to develop new intervention strategies for control of cystic echinococcosis.

overall_design: Examination of small RNA populations in 3 different life stages of Echinococcus granulosus

first: Shengyue

last: Wang

first: Wenbao

last: Zhang


title: The inactive X chromosome is epigenetically unstable and transcriptionally labile in breast cancer

city: Paris

first: Ronan

last: Chaligné

address: 26, Rue d'Ulm

institute: Institut Curie

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

overall_design: Refer to individual Series

last: Chaligne

first: Tatiana

last: Popova

first: Marco-Antonio

last: Mendoza-Parra

first: Mohamed-Ashick

last: Saleem

first: David

last: Gentien

first: Kristen

last: Ban

first: Tristan

last: Piolot

first: Olivier

last: Leroy

first: Odette

last: Mariani

first: Hinrich

last: Gronemeyer

first: Anne

last: Vincent-Salomon

first: Marc-Henri

last: Stern

first: Edith

last: Heard


title: Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome

status: Public on Jan 15 2015

city: Singapore

first: Henry

last: Yang

address: 28 Medical Drive

country: Singapore

institute: Cancer Science Institute of Singapore

summary: Somatic mutations in the spliceosome gene ZRSR2— located on the X chromosome — are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3΄ splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here, we characterize ZRSR2 as an essential component of the minor spliceosome (U12-dependent) assembly. shRNA mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns, and RNA-Sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns while splicing of the U2-type introns remain mostly unaffected. ZRSR2 deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS.

overall_design: RNA sequencing was performed on 16 bone marrow samples (MDS and normal)  and six samples of control or ZRSR2 shRNA transduced TF-1 cells and data was analysed for aberrant splicing caused by ZRSR2 mutations/deficiency.

first: Vikas

last: Madan

first: Deepika

last: Kanojia

first: Jia

last: Li

first: Ryoko

last: Okamoto

first: Aiko

last: Sato-Otsubo

first: Alexander

last: Kohlmann

first: Masashi

last: Sanada

first: Vera

last: Grossmann

first: Janani

last: Sundaresan

first: Yuichi

last: Shiraishi

first: Satoru

last: Miyano

first: Felicitas

last: Thol

first: Arnold

last: Ganser

first: Torsten

last: Haferlach

first: Seishi

last: Ogawa

first: H

last: Koeffler


title: Transcriptomics of cold stress in alligatorweed (Alternanthera philoxeroides)

city: Jinan

first: Liu

last: Dasheng

state: Shandong

address: Lishan Lu 50

institute: Shandong Institute of Environmental Science

summary: Research conducted, including the rationale: Alligatorweed is an invasive perennial primarily aquatic weed that can also grow terrestrially and thus has potential to become problematic in agricultural settings. It is invasive on at least three continents. Despite its invasiveness, less than 100 genes have been sequenced from this species. Alligatorweed has recently increased its range northwards in China, and it is unknown if the range expansion has a genetic or epigenetic basis. We have used a next generation RNA sequencing approach to examine gene expression in a three individuals from the northern edge of its range and from the central portion of its range in China to look for different responses to cold temperatures using a common garden approach. We identified numerous differences in gene expression associated with the cold response between these two populations. Gene set and sub-network enrichment analysis indicated differences in the response of photosynthetic processes and oxidative stress responses were different between the two populations. However, alterations in genes controlling the C-REPEAT/DRE BINDING FACTOR (CBF) regulon were not indicated. Additionally, we assembled over 75,000 genes of which over 65,000 had long open reading frames with similarity to sequences from arabidopsis. Single nucleotide polymorphisms unique to the northern and southern individuals were identified, and several of these were in genes such as RESPONSIVE TO DEHYDRATION 21 that could impact the cold response of the plants.

overall_design: Samples were collected from cold-treated plants from each populations  (The "central" populations collected in Shanghai and "Northern" population collected in Jinan,Shandong in China) with three biological replicates from each population.

first: David

last: Horvath


title: Transcriptome profiling of untreated and doxycycline-treated HeLa/TO-SmN

city: ChiaYi

first: Chin

address: 168 University Road, Minhsiung

department: Life Science

institute: National Chung Cheng University

summary: Purpose is to examine the effect of SmN expression to gene expression in HeLa

overall_design: Compare the differences between the transcriptome of untreated and doxycycline-treated HeLa/TO-SmN in which expression of SmN is under doxycycline control


title: Msi2 sustains the MLL leukemia stem cell regulatory program

city: New York

first: Yuheng

last: Lu

address: 417 East 68th Street

institute: Memorial Sloan Kettering Cancer Center

summary: Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Genetic and epigenetic alterations cause a dysregulated developmental program in leukemia. The MSI2 RNA binding protein has been previously shown to predict poor survival in leukemia. We demonstrate that the conditional deletion of Msi2 results in delayed leukemogenesis, reduced disease burden and a loss of LSC function. Gene expression profiling of the Msi2 ablated LSCs demonstrates a loss of the HSC/LSC and an increase in the differentiation program. The gene signature from the Msi2 deleted LSCs correlates with survival in AML patients. MSI2’s maintains the MLL self-renewal program by interacting with and retaining efficient translation of Hoxa9, Myc and Ikzf2. We further demonstrate that shRNA depletion of the MLL target gene Ikzf2 also contributes to MLL leukemia cell survival. Our data provides evidence that MSI2 controls efficient translation of the oncogenic LSC self-renewal program and a rationale for clinically targeting MSI2 in myeloid leukemia.

overall_design: RNA-Seq was performed on sorted c-Kit high leukemic cells from 2 Msi2 -/- and 2 Msi2 f/f mice.

first: Michael

middle: G

last: Kharas


title: RNA sequencing (RNA-SEQ) of EPAS1 knockdown by siRNA in endothelial cells

first: Seungyeul

last: Yoo

address: 1425 Madison Avenue

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

summary:
Purpose: By integrating DNA methylation and gene expression of COPD lung tissues, we identified EPAS1 as a key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profiles and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system developement. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human and mouse endothelial cells HUVEC and C166. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes.
Methods: The cell lines of HUVEC (Lonza, MD, USA) and C166 (American Type Culture Collection, VA, USA) were cultured in the appropriate media at 37°C with 5% CO2. The cells were transfected with EPAS1 siRNA and non-targeting negative control siRNA (Life Technologies, CA, USA) using Lipofectamine RNAiMAX as recommended transfection protocols by the manufacturer. After the treatments with 5nM Silencer® Select siRNA (s4700 for EPAS1, s65525 for Epas1; Life Technologies, USA) for 48 hours, the total RNA was purified with RNeasy Mini Kit (QIAGEN, Germany). The efficiencies of knocked down the EPAS1 expression were assessed by qPCR with 1.4% for HUVEC, 3.2% for C166. Approximately 250 ng of total RNA per sample were used for library construction by the TruSeq RNA Sample Prep Kit (Illumina) and sequenced using the Illumina HiSeq 2500 instrument with 100nt single read setting according to the manufacturer's instructions. Sequence reads were aligned to human genome assembly hg19 and mouse genome assembly mm10, respectively, using Tophat [96]. Total 23,228 human and 22,609 mouse genes were quantified using Cufflinks [96]. siRNA signatures were derived by comparing expression profiles of EPAS1 or Epas1 siRNAs with non-targeting siRNAs at paired t-test p-value cutoff 0.05 with resulting signature sizes of 2,796 and 3,730, and corresponding q-values [97] 0.11 and 0.07 for HUVEC and C166, respectively.
Results: When comparing endothelial cells treated with EPAS1 siRNAs and scrambled siRNAs, we identified an EPAS1 siRNA signature consisting of 2796 and 3730 genes in human and mouse endothelial cell lines, respectively, whose expression levels significantly changed (t-test p-value<0.05), including EPAS1 itself (p-value = 0.002 and 0.02) and the EPAS1 downstream target gene VEGFA (p-value = 0.03 and 0.01). The EPAS1 siRNA signatures derived from human and mouse cell lines were highly consistent, with 695 genes in common to both signatures (p-value = 7.2x10-65). Both signatures not only significantly overlapped with EPAS1 downstream genes (p-value = 7.3x10-7 and 1.5x10-12), but also with hypoxia response genes in endothelial cells (Fisher’s Exact Test p-value = 5.8x10-8 and 1.2x10-12 in the human and mouse signatures, respectively). Moreover, the EPAS1 siRNA signatures consistently overlapped genes associated with the COPD severity phenotypes. These results together validate that EPAS1 causally regulates the downstream target genes we predicted, and that these genes in turn affect COPD development and progression.

overall_design: For each of human and mouse cell lines,  3 siRNA control cells, and 3 siRNA EPAS1 knockdown cells were used and analyzed. To identify EPAS1 signatures, paired t-test was performed between control siRNA and EPAS1 siRNA samples.

first: Sachiko

last: Takikawa

first: Charles

last: Powell

first: Jun

last: Zhu


title: Genome-wide mapping of transcription start sites in a ∆set2 strain

status: Public on Jan 14 2015

city: Solna

first: Vicent

last: Pelechano

address: Nobels väg 16

department: MTC

institute: ScilifeLab - Karolinska Institutet

summary: Here we quantified the transcription start site usage in a WT strain (BY4741) and a ∆set2 strain associated with the appearence of cryptic transcription start sites.

overall_design: Transcription start site usage was quantified using the 5’cap sequencing aproach for S. cerevisiae strains. Biological duplicates were included.

first: Wu

last: Wei

first: Lars

last: Steinmetz


title: Dnmt3L-histone H3 binding is necessary for male fertility and non-CG methylation

city: Athens

first: Robert

last: Schmitz

address: B416 Davison Life Sciences

department: Genetics

institute: University of Georgia

summary: Patterns of DNA methylation are established during gametogenesis. The catalytically-inactive adaptor Dnmt3L is crucial to ensure this occurs correctly. In vitro, Dnmt3L binds to the N terminal tail of histone H3 but the function of this interaction during development is unknown. Here we show that Dnmt3L-histone H3 interaction is necessary for spermatogenesis. Mutant animals exhibit reduced fertility, defective methylation establishment at retrotransposons coupled with their reactivation and meiotic catastrophe. The spermatogonial stem cell pool is also defective, with mutant cells displaying marked changes in gene expression. Genome-wide methylation analysis reveals reductions in CG methylation as well as severe loss of non-CG methylation suggesting that non-CG methylation is specifically sensitive to the ability of Dnmt3L to bind histone H3.

overall_design: MethylC-Seq of wild-type and Dnmt3LA/A 1dpp prospermatagonia

first: Steen

last: Ooi


title: Enhancer Sequence Variants and Transcription Factor Deregulation Synergize to Construct Pathogenic Regulatory Circuits in B Cell Lymphoma (RNA-Seq)

city: St. Louis

first: Jacqueline

last: Payton

address: 660 S. Euclid Ave

department: Pathology and Immunology

institute: Washington University School of Medicine

summary: Most B cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC-B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions transcriptional circuits from normal GC-B cells and commandeers enhancers from other lineages. Moreover, independent sets of transcription factors, whose expression is deregulated in FL, target commandeered versus decommissioned REs. Our approach reveals two distinct subtypes of low-grade FL, whose pathogenic circuitries resemble GC-B or activated B cells. Remarkably, FL-altered enhancers also are enriched for sequence variants, including somatic mutations, which disrupt transcription factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC-B transformation.

overall_design: Expression profiles of follicular lymphoma, centrocyte and peripheral blood B cells were generated by deep sequencing, using Illumina Hi-Seq 2000.

first: Eugene

last: Oltz

first: Olivia

middle: I

last: Koues


title: Enhancer Sequence Variants and Transcription Factor Deregulation Synergize to Construct Pathogenic Regulatory Circuits in B Cell Lymphoma (ChIP-Seq, FAIRE-Seq)

overall_design: Molecular profiling of follicular lymphoma, resting peripheral blood and tonsillar B cells using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) and chromatin immunoprecipitation (H3ac and H3K27ac).


title: Transcriptional response to UV treatment in several Arabidopsis thaliana accessions

status: Public on Jan 13 2015

city: Cologne

first: Ales

last: Pecinka

address: Carl von Linne Weg 10

institute: Max Planck Institute for Plant Breeding Research

summary: UV radiation is a ubiquitous component of solar radiation that affects plant growth and development. Analysis of natural variation in response to UV radiation revealed significant differences among natural accessions of Arabidopsis thaliana. However, the genetic basis of this is to a large extent unknown. Here, we analyzed the response of Arabidopsis accessions to UV radiation stress by performing RNA-sequencing of three UV sensitive and three UV resistant accessions. The genome-wide transcriptional analysis revealed a large number of genes significantly up- or down-regulated only in sensitive or only in resistant accessions, respectively. Mutant analysis of few selected candidate genes suggested by the RNA-sequencing results indicate a connection between UV radiation stress and plant-pathogen-like defense responses.

overall_design: Examination of transcriptional changes in response to UV treatment in Arabidopsis natural accessions

first: Thomas

last: Piofczyk

first: Ganga

last: Jeena