title: Small RNA Solexa sequencing of colorectal tumor and adjancent normal tissues

city: Kunming

first: Yun

last: Zheng

state: Yunnan

address: 452 Fengyuan Road

institute: Yunnan Agricultural University

summary: A pair of stage III colorectal cancer (CRC) tumor and adjacent normal tissue were collected from a patient without Transcatheter Arterial Infusion chemotherapy (TAI) during surgical resection of CRC tumors. Another pair of stage III CRC tumor and adjancent normal tissue were collected from another patient who had been treated with TAI one week before surgical resection. Total RNA of the collected tissues were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The integrity of the RNA was checked with an ultraviolet spectrophotometry and 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). Construction of small RNA libraries from size fractionated RNA was carried out by following Solexa protocol and the obtained libraries were sequenced by Illumina HiSeq 2000 sequencer. We identified some de-regulated miRNAs in CRC tumor tissues. The expression levels of miR-142-5p were significantly reduced in tumor tissues of stage III CRC, then were increased significantly in tumor tissues receiving TAI and higher than tumor tissues without TAI. miR-142-5p is a potential tumor suppressor in CRC and is upregulated in tumor tissues after TAI, suggesting its potential clinical values for testing the functionality of TAI and predicting progress of CRC.

overall_design: Examination of small RNA transcriptomes in colorectal tumor and adjancent normal tissues without and with Transcatheter Arterial Infusion chemotherapy using Illumina HiSeq2000 sequencer.


title: Single-cell RNA-Seq reveals dynamic, random monoallelic gene expression in mammalian cells

status: Public on Jan 10 2014

city: Stockholm

first: Daniel

last: Ramsköld

address: Nobels väg 3

country: Sweden

institute: Karolinska Institutet

type: Expression profiling by high throughput sequencing

summary: In the diploid genome, genes come in two copies, which can have different DNA sequence and where one is maternal and one is paternal. In a particular cell, a gene could potentially be expressed from both copies (biallelic expression) or only one (monoallelic). We performed RNA-Sequencing on individual cells, from zygote to the cells of the late blastocyst, and also individual cells from the adult liver. Using first generation crosses between two distantly related mouse strains, CAST/Ei and C57BL/6, we determined the expression separately from the maternal and paternal alleles. We found that half of the genes were expressed by only one allele, randomly so that some cells would express the paternal allele, some the maternal and a few cell both alleles. We also observed the spread of the progressive inactivation of the paternal X chromosome.

overall_design: First generation mouse strain crosses were used to study monoallelic expression on the single cell level

first: Qiaolin

last: Deng

first: Björn

last: Reinius

first: Rickard

last: Sandberg


title: The Demethylase JMJD2C/KDM4C Localizes to H3K4me3 Positive Transcription Start Sites (ChIP-seq ESCs)

status: Public on Jan 09 2014

city: Copenhagen N

first: Marianne

middle: Terndrup

last: Pedersen

address: Ole Maaloes Vej 5

department: University of Copenhagen

country: Denmark

institute: BRIC

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We have mapped binding sites for the histone demethylase, Jmjd2c/Kdm4c/Gasc1, in mouse embryonic stem cells (ESCs).

overall_design: ChIP-seq was performed using an antibody recognizing Jmjd2c. Chromatin was obtained from conditional Jmjd2c knockout ESCs cultured in the absence or presence of OHT to induce activation of Cre recombinase and loss of Jmjd2c expression.

middle: T

first: Anne

last: Laugesen


title: Small RNA Profiling of Murine Adult Stomach

city: Reno

first: Nicole

last: Ortogero

state: Nevada

address: 1664 N. Virginia St

department: Physiology and Cell Biology

country: USA

institute: University of Nevada, Reno

type: Non-coding RNA profiling by high throughput sequencing

summary: Murine Adult Stomach Small RNA

overall_design: Stomach samples were collected from adult mice.  Small RNA was isolated from samples and cDNA libraries were generated for PGM sequencing.  Sequences were aligned with Torrent Server tMap.

first: Grant

last: Hennig

first: Dickson

last: Luong

first: Bhupal

last: Bhetwal

first: Seungil

last: Ro

first: John

last: McCarrey

first: Wei

last: Yan


title: The Demethylase JMJD2C/KDM4C Localizes to H3K4me3 Positive Transcription Start Sites (ChIP-seq KYSE150 cell line)

summary: We have mapped binding sites for the histone demethylase, JMJD2C/KDM4C/GASC1, and the effect of JMJD2C depletion on H3K9me3 and H3K36me3 distributions in KYSE150 cells.  The human esophageal carcinoma cell line, KYSE150, contains an amplification of the JMJD2C locus.

overall_design: ChIP-seq was performed using chromatin from control or JMJD2C-depleted KYSE150 cells and antibodies recognizing JMJD2C, H3K4me3, H3K9me3 or H3K36me3.


title: Context Dependency of Set1/COMPASS Mediated H3 Lysine 4 Trimethylation

city: Kansas City

first: Malcolm

last: Cook

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

state: MO

address: 1000 E 50th Street

department: Big Data AI

institute: Stowers Institute For Medical Research

summary: The stimulation of trimethylation of histone H3 lysine 4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied with multiple mechanisms proposed for this form of histone crosstalk. Cps35/Swd2 within COMPASS is considered to bridge these processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation without interacting with Cps35/Swd2, and such crosstalk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we use biochemical, structural, in vivo, and ChIP-seq approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the crosstalk.  Furthermore, the apparent wild-type levels of H3K4 trimethylation (H3K4me3) in the 762-Set1 strain is due to rogue methylase activity of this mutant resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to gene bodies and intergenic regions. We have also performed detailed screens and identified yeast strains lacking H2Bub, but containing intact H2Bub enzymes, that have normal levels of H3K4me3, suggesting that ubiquitination may not directly stimulate COMPASS, but rather works in a context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the ubiquitination machinery and Cps35/Swd2 function to focus COMPASS’ H3K4me3 activity at promoter-proximal regions in a context dependent manner.

overall_design: ChIP-Seq for H3K4ME3 in S. cerevisie wild-type strains and strains expressing a truncated form of Set1: aa762-1080 Set1.  H3K4ME3 ChIP-Seq was also compared for wild-type,  leo1 knockout, and chd1 knockout strains

first: Janet

middle: L

last: Thornton

first: Xin

last: Gao

first: Ali

last: Shilatifard


title: The Demethylase JMJD2C/KDM4C Localizes to H3K4me3 Positive Transcription Start Sites (ChIP-seq MEFs)

summary: We have mapped binding sites for the histone demethylase, Jmjd2c/Kdm4c/Gasc1, in mouse embryonic fibroblasts (MEFs) and the impact of Jmjd2c depletion on H3K9me3 and H3K36me3 distributions.

overall_design: ChIP-seq was performed using antibodies recognizing Jmjd2c, H3K9me3 or H3K36me3. Chromatin was obtained from conditional Jmjd2c knockout MEFs cultured in the absence or presence of OHT to induce activation of Cre recombinase and loss of Jmjd2c expression.


title: Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing

city: New Delhi

first: Amar

last: Kumar

state: Delhi

address: Chatra Marg

department: Botany

country: India

institute: University of Delhi

summary:
Purpose: MicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing.
Results: Small RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAIRY MERISTEM (HAM) respectively, were experimentally validated using 5′RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions.
Conclusions: We report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber developmental process.

overall_design: Total seven (Leaf, Root, Stem, Potato Tuber stage 0(PT0),Potato Tuber stage 1(PT1),Potato Tuber stage 2(PT2),Potato Tuber stage 3(PT3) ) small RNA libraries were consctructed and sequenced by deep sequencing  using Illumina GAIIx.

first: Nisha

last: Lakhotia

first: Gopal

last: Joshi

first: Ankur

middle: R

last: Bhardwaj

first: Surekha

last: Katiyar-Agarwal

first: Manu

last: Agarwal

first: Arun

last: Jagannath

first: Shailendra

last: Goel


title: CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells.

status: Public on Jan 08 2014

city: Barcelona

first: Miguel

last: Beato

address: Aiguader 88

country: Spain

institute: CRG

summary: Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer.

overall_design: PARP-1 activation mechanism by CDK2 in response of progestin in breast cancer cells

first: Roni

middle: G

last: Wright

first: Giancarlo

last: Castellano

first: Jaume

last: Bonet

first: François

last: Le Dilly

first: Jofre

last: Font-Mateu

first: Cecilia

last: Ballaré

first: A

middle: S

last: Nacht

first: Daniel

last: Soronellas

first: Baldo

last: Oliva


title: Genome-wide analysis of K3K27ac, H2AZ and methylation data in mouse embryonic stem cells

city: Urbana

first: Chieh-Chun

last: Chen

state: Illinois

address: 1206 West Gregory Drive

department: Bioengineering

institute: University of illinois

type: Methylation profiling by high throughput sequencing

summary: We generated two types 5-methylcytosine (5-mC) data in E14 mouse embryonic stem cells, using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) and DNA digestion by methyl-sensitive restriction enzymes followed by sequencing (MRE-seq).

overall_design: Examination of 2 histone modifications and methylation in mouse embryonic stem cells

first: Shu

last: Xiao

first: Xiaoyun

last: Xing

first: Chunxiao

last: Song

first: Dan

last: Xie

first: Xiaoyi

last: Xao

first: Darina

last: McDee

first: Ting

last: Wang

first: Chuang

last: He

first: Shang

last: Zhong


title: RNA-sequencing of the brain transcriptome implicates dysregulation of neuroplasticity, circadian rhythms, and GTPase binding in bipolar disorder

city: Bethesda

first: Nirmala

last: Akula

state: Maryland

address: Building 35, Room 1A-205

department: Human Genetics Branch

institute: National Institute of Mental Health/National Institutes of Health

summary: See "Akula et al., Molecular Psychiatry in Press". RNA-sequencing (RNA-seq) is a powerful technique to investigate the complexity of gene expression in the human brain. We used RNA-seq to survey the brain transcriptome in high-quality post-mortem dorsolateral prefrontal cortex from 11 individuals diagnosed with bipolar disorder (BD) and from 11 age- and gender-matched controls.  Deep sequencing was performed, with over 350 million reads per specimen. At a false-discovery rate of <5%, we detected 5 differentially-expressed (DE) genes and 12 DE transcripts, most of which have not been previously implicated in BD.  Among these, PROM1/CD133 and ABCG2 play important roles in neuroplasticity. We also show for the first time differential expression of long non-coding RNAs (lncRNAs) in BD.  DE transcripts include those of SRSF5 and RFX4, which along with lncRNAs play a role in mammalian circadian rhythms. The DE genes were significantly enriched for several Gene Ontology (GO) categories. Of these, genes involved with GTPase binding were also enriched for BD-associated SNPs from previous genome-wide association studies, suggesting that differential expression of these genes is not simply a consequence of BD or its treatment.  Many of these findings were replicated by microarray in an independent sample of 60 cases and controls. These results highlight common pathways for inherited and non-inherited influences on disease risk that may constitute good targets for novel therapies.

overall_design: Brain transcriptome in bipolar disorder

first: Francis

middle: J

last: McMahon


title: The Demethylase JMJD2C/KDM4C Localizes to H3K4me3 Positive Transcription Start Sites

first: Marianne

last: Pedersen

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Small RNA expression throughout the development of Drosophila virilis

status: Public on Jan 13 2014

city: Pasadena

first: Maria

last: Ninova

state: CA

address: 1200 E California Blvd

department: Division of Biology and Biological Engineering

institute: Calfironia Institute of Technology

summary: Examination of temporal small RNA expression during the embryonic development, larval and adult stages of Drosophila virilis

overall_design: Small RNA sequencing of samples representing different stages of D. virilis development, including eight 2-hour intervals during the first 16 hours of development (0-2h, 2-4h, etc.), late embryos until hatching (16-30h), 3rd instar larvae and adults

first: Matthew

last: Ronshaugen

first: Sam

last: Griffiths-Jones


title: Sox4 is a key oncogenic target in C/EBPa mutant Acute Myeloid Leukemia

city: Birmingham

first: Salam

middle: Adli

last: Assi

address: IBR

department: Institute for Cancer and Genomic Sciences

country: United Kingdom

institute: University of Birmingham

summary: Mutation or epigenetic silencing of the transcription factor C/EBPa is observed in ~10% of patients with acute myeloid leukemia (AML). In both cases, a common global gene expression profile is observed, but down-stream targets relevant for leukemogenesis are not known. Here we identify Sox4 as a direct target of C/EBPa whereby its expression is inversely correlated with C/EBPa activity. Downregulation of Sox4 abrogated increased self-renewal of leukemic cells and restored their differentiation. Gene expression profiles of leukemia initiating cells (LICs) from both Sox4 overexpression and murine mutant C/EBPa AML models clustered together, but differed from other types of AML. Our data demonstrate that Sox4 overexpression resulting from C/EBPa inactivation contributes to the development of leukemias with a distinct LIC phenotype.

overall_design: A ChIP-seq sample of C/EBPa in Macrophages is used in this study

first: Hong

last: Zhang

first: Min

last: Ye

first: Meritxell

last: Alberich-Jordà

first: Giovanni

last: Amabile

first: Henry

last: Yang

first: Philipp

middle: B

last: Staber

first: Annalisa

last: DiRuscio

first: Rob

last: Welner

first: Alexander

last: Ebralidze

first: Junyan

first: Elena

last: Levantini

first: Véronique

last: Lefebvre

first: Peter

last: Valk

first: Ruud

last: Delwel

first: Maarten

last: Hoogenkamp

first: Claus

last: Nerlov

first: Jörg

last: Cammenga

first: Borja

last: Saez

first: David

last: Scadden

first: Constanze

last: Bonifer

first: Daniel

last: Tenen


title: Analysis of the mRNA Targetome of MicroRNAs Expressed by Marek’s Disease Virus

status: Public on Jan 07 2014

city: Durham

first: Oren

last: Parnas

state: NC

address: 430 CARL, Research Dr.

department: MGM

institute: Duke university

summary: Marek’s disease virus 1 (MDV-1), an oncogenic -herpesvirus that induces T-cell lymphomas in chickens, serves as  model system to study transformation by lymphotropic herpesviruses. Like the oncogenic human  -herpesviruses Kaposi’s  sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), MDV-1 encodes several viral microRNAs (miRNAs). One  MDV-1 miRNA, miR-M4, shares the same “seed” targeting sequence with both a KSHV miRNA, miR-K11, and cellular miR-155.  Importantly, miR-M4 plays a critical role in T-cell transformation by MDV-1, while miR-K11 and cellular miR-155 are thought  to play key roles in B-cell transformation by KSHV and EBV, respectively. Here, we present an analysis of the mRNAs targeted by  viral miRNAs expressed in the chicken T-cell line MSB1, which is naturally coinfected with MDV-1 and the related nonpathogenic  virus MDV-2. Our analysis identified>1,000 endogenous mRNAs targeted by miRNAs encoded by each virus, many of  which are targeted by both MDV-1 and MDV-2 miRNAs. We present a functional analysis of an MDV-1 gene, RLORF8, targeted  by four MDV-1 miRNAs and a cellular gene, encoding interleukin-18 (IL-18) and targeted by both MDV-1 and MDV-2 miRNAs,  and show that ectopic expression of either protein in a form resistant to miRNA inhibition results in inhibition of cell proliferation.  Finally, we present a restricted list of 9 genes targeted by not only MDV-1 miR-M4 but also KSHV miR-K11 and human  miR-155. Given the critical role played by miR-155 seed family members in lymphomagenesis in humans and chickens, these  mRNA targets may contain genes whose inhibition plays a conserved role in herpesvirus transformation.

overall_design: PAR-CLIP experiment of MSB1 cells

first: Bryan

last: Cullen


title: Identification of genes critical for resistance to infection by West Nile virus using RNA-Seq analysis

city: New Haven

first: Ruth

last: Montgomery

state: CT

address: 300 Cedar St.

department: Internal Medicine

institute: Yale University School of Medicine

summary:
Background: West Nile virus is an emerging infection of biodefense concern and there are no available treatments or vaccines. Here we used a high-throughput method based on a novel gene expression analysis, RNA-Seq, to give a global picture of differential gene expression by primary human macrophages of 10 healthy donors infected in vitro with WNV.

Results: From a total of 50 million reads per sample, we employed a Bayesian hierarchical mixture model to identify 4,026 transcripts that were differentially expressed after infection. Both predicted and novel gene changes were detected, as were gene isoforms, and while many of the genes were expressed by all donors, some were unique. Knock-down of genes not previously known to be associated with WNV resistance identified their critical role in control of viral infection.

Conclusions: Our study distinguishes both common gene pathways as well as novel cellular responses. Such analysis will be valuable for translational studies of susceptible and resistant individuals -- and for targeting therapeutics -- in multiple biological settings.

overall_design: Differential gene expression by primary human macrophages of 10 healthy donors infected in vitro with WNV were generated by RNA-Seq.


title: Whole genome transcription profiling of the L5 spinal nerve transection model of neuropathic pain in the rat, at different sequencing depths

city: Malaga

first: James

last: Perkins

address: Pl. Hosp. Civil

department: Hospital Civil

institute: Hospital Carlos Haya


title: Whole genome transcription profiling of the L5 spinal nerve transection model of neuropathic pain in the rat, at different sequencing depths (RNA-Seq)

summary: Poly(A) enriched RNA derived from the L5 DRG 7 days following L5-SNT and from naïve L5-DRG tissue was subjected to RNA-seq analysis at different sequencing depths

overall_design: 6 biological replicates (3 case – SNT subjected L5-DRG tissue, 3 control – naïve L5-DRG tissue). Each biological replicate was divided B46into 3 technical replicates; each of the technical replicates for a given sample was sequenced to a depth of 17M, 25M or 50M reads. Reads were single stranded and 34bps in length. Multiplexing was used in order to generate the read depths of different sizes. The gene expression values and fold changes in expression between naive and SNT samples were compared to those generated by a microarray experiment carried out on further technical replicates of the same samples, details in the manuscript (submitted - under revision).

first: Ana

last: Antunes-Martins

first: Margarita

last: Calvo

first: John

last: Grist

first: Werner

last: Rust

first: Ramona

last: Schmid

first: Tobias

last: Hildebrandt

first: Matthias

last: Kohl

first: Christine

last: Orengo

first: Stephen

last: McMahon

last: Bennett


title: Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere

city: Edinburgh

first: Alastair

middle: Robert

last: Kerr

state: Midlothian

address: 2.21 Michael Swann Building, Kings Buildings, Mayfield Road

department: Wellcome Centre for Cell Biology

institute: University of Edinburgh

summary: To protect against aneuploidy, chromosomes must attach to microtubules from opposite poles (“biorientation”) prior to their segregation during mitosis. Biorientation relies on the correction of erroneous attachments by the aurora B kinase, which destabilizes kinetochore-microtubule attachments that lack tension. Incorrect attachments are also avoided because sister kinetochores are intrinsically biased towards capture by microtubules from opposite poles. Here we show that shugoshin acts as a pericentromeric adaptor that plays dual roles in biorientation in budding yeast. Shugoshin maintains the aurora B kinase at kinetochores that lack tension, thereby engaging the error correction machinery. Shugoshin also recruits the chromosome-organising complex, condensin, to the pericentromere. Pericentromeric condensin biases sister kinetochores towards capture by microtubules from opposite poles. Overall, shugoshin integrates a bias to sister kinetochore capture with error correction to enable chromosome biorientation. Our findings uncover the molecular basis of the bias to sister kinetochore capture and expose shugoshin as a pericentromeric hub controlling chromosome biorientation.

overall_design:
Two experiments:
Experiment A: Sgo1 is required for condensin localization in the pericentromere. Sample 1: Wild type input DNA Sample 2: Wild type Brn1-6HA ChIP DNA, Sample 3 sgo1D input DNA, Sample 4 sgo1D Brn1-6HA ChIP DNA; Experiment B: Sgo1 is not required for cohesin localization in the periecentromere: Sample 5: wild type input DNA, Sample 6 Wild type Scc1-6HA ChIP DNA, Sample 7, sgo1D input DNA, Sample 8 sgo1D Scc1-6HA ChIP DNA. 1 replicate of each repeat

first: Kitty

last: Verzijlbergen

first: Adele

last: Marston


title: Single-base resolution DNA methylomes of soybean recombinant inbred lines reveals heritability patterns of natural methylation variants (RNA-seq)

city: La Jolla

first: Joseph

last: Ecker

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

summary: Microbe associated molecular pattern (MAMP)-triggered immunity (MTI) is an important component of the plant innate immunity response to invading pathogens. Although several MTI responses can be measured in different plant species, their magnitude is likely plant-species specific and even cultivar specific. In this work, we show that the variation in gene expression, either under untreated or treated conditions, is inherited. In addition, genes with potential additive and non-additive effects were identified in two mapping lines, several of these with a potential function in the control of the innate immunity. Likewise, we observed that some genes differentially expressed across the parental and mapping lines showed different DNA methylation patterns. Finally, a gene regulatory module analysis identified key networks that likely control soybean innate immunity. The data presented represent the basis for further functional analysis that can lead to a better understanding of the soybean innate immunity response.

overall_design: RNA-Seq data collected from Glycine max leaves treated with MAMPs of two parental lines of a RIL population and two individual RILs.

first: Robert

last: Schmitz


title: Adult stem cells in the small intestine are intrinsically programmed with their location-specific function

city: Utrecht

first: Michal

last: Mokry

address: Lundlaan 6

country: Netherlands

institute: Wilhelmina Children's Hospital, University Medical Center Utrecht

summary: Differentiation and specialisation of epithelial cells in the small intestine is regulated in two ways. First, there is differentiation along the crypt-villus axis of the intestinal stem cells into absorptive enterocytes, Paneth, goblet, tuft, enteroendocrine or M-cells, which is mainly regulated by WNT. Second, there is specialization along the cephalocaudal axis with different absorptive and digestive functions in duodenum, jejunum and ileum that is controlled by several transcription factors such as GATA4. However, so far it is unknown whether location-specific functional properties are intrinsically programmed within stem cells or if continuous signalling from mesenchymal cells is necessary to maintain the location-specific identity of the small intestine.   By using the pure epithelial organoid technique, we show that region-specific gene expression profiles are conserved throughout long-term cultures of both mouse and human intestinal stem cells and correlated with differential Gata4 expression. Furthermore, the human organoid culture system demonstrates that Gata4-regulated gene expression is only allowed in absence of WNT signalling.   These data show that location-specific function is intrinsically programmed in the adult stem cells of the small intestine and that their differentiation fate is independent of location-specific extracellular signals.  In light of the potential future clinical application of small intestine-derived organoids, our data imply that it is important to generate GATA4-positive and GATA4-negative cultures to regenerate all essential functions of the small intestine.

overall_design: RNA sequencing of intestinal crypts, villi and cultured organoids derived from mouse duodenum, jejunum and ileum


title: Gene Model 129 (Gm129) Encodes a Novel Transcriptional Repressor that Modulates Circadian Gene Expression

city: Chapel Hill

first: Rui

last: Ye

address: 120 Mason Farm road, Genetic medicine building, suite 3010

department: Biochemistry and Biophysics

institute: UNC Chapel Hill

summary: The mammalian circadian clock is a molecular oscillator composed of a feedback loop that involves transcriptional activators CLOCK and BMAL1, and repressors Cryptochrome (CRY) and Period (PER). Here we show that a direct CLOCK-BMAL1 target gene, Gm129, is a novel regulator of the feedback loop. ChIP analysis revealed that the CLOCK:BMAL1:CRY1 complex strongly occupies the promoter region of Gm129. Both mRNA and protein levels of GM129 exhibit high amplitude circadian oscillations in mouse liver, and Gm129 gene encodes a nuclear-localized protein that directly interacts with BMAL1 and represses CLOCK:BMAL1 activity. In vitro and in vivo protein-DNA interaction results demonstrate that, like CRY1, GM129 functions as a repressor by binding to the CLOCK:BMAL1 complex on DNA. Although Gm129-/- or Cry1-/- Gm129-/- mice retain a robust circadian rhythm, the peaks of Nr1d1 and Dbp mRNAs in liver exhibit significant phase delay compared to control. Our results suggest that, in addition to CRYs and PERs, GM129 protein contributes to the transcriptional feedback loop by modulating CLOCK:BMAL1 activity as a transcriptional repressor.

overall_design: Examination of 3 transcriptional regulators in mouse liver

first: Yunus

last: Annayev

first: Sheera

last: Adar

first: Yi-Ying

last: Chiou

first: Jason

last: Lieb

first: Aziz

last: Sancar


title: Comparison of KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes

city: Vienna

first: Shuan

last: Rao

address: Dr. Bohrgasse 7

country: Austria

institute: IMBA

summary: Primary pneumocytes from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates were cultured for 48 hours and infected with AdCre-GFP to induce expression of the KrasG12D oncogene and concomitant Atg5 deletion. The transcriptional profile of those cells was determined by mRNA sequencing and uncovered differential expression in cellular movement, inflammatory response and oxidative stress response.

overall_design: Comparison of transcriptomes from KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes

first: S

first: J

last: Penninger


title: RNA-seq differential expression studies: more sequence, or more replication?

city: Chicago

first: Jie

last: Zhou

state: ILLINOIS

address: 900 East 57th Street, KCBD 10100

institute: University of Chicago IGSB

summary: Motivation: RNA-seq is replacing microarrays as the primary tool for gene expression studies. Many RNA-seq studies have used insufficient biological replicates, resulting in low statistical power and inefficient use of sequencing resources. Results: We show the explicit trade-off between more biological replicates and deeper sequencing in increasing power to detect differentially expressed (DE) genes. In the human cell line MCF-7, adding more sequencing depth after 10M reads gives diminishing returns on power to detect DE genes, while adding biological replicates improves power significantly regardless of sequencing depth. We also propose a cost-effectiveness metric for guiding the design of large scale RNA-seq DE studies. Our analysis showed that sequencing less reads and perform more biological replication is an effective strategy to increase power and accuracy in large scale differential expression RNA-seq studies, and provided new insights into efficient experiment design of RNA-seq studies

overall_design: Treatment (10nM E2 treatment for 24h) and control MCF7 cells are both replicated 7 times, and collected for mRNA-seq. Reads are then subsampled for statistical analysis.

first: Yuwen

last: Liu

first: Kevin

last: White


title: Evolutionary and ontogenetic changes in RNA editing in human, chimpanzee and macaque brains

city: Shang Hai

first: zs

last: li

address: No. 320, Yueyang Road

country: China

institute: Partner Institute of Computational Biology

summary: we used RNA-Seq to quantify the RNA editing level at more than 8,000 previously annotated exonic A-to-I RNA editing sites in two brain regions - prefrontal cortex and cerebellum - of humans, chimpanzees and rhesus macaques. We observed substantial conservation of RNA editing levels between the brain regions, as well as among the three primate species. Evolutionary changes in RNA editing were nonetheless evident among the species. Across lifespan, we observed an increase of the RNA editing level with advanced age in both brain regions of all three primate species.

sample_organism:
Macaca mulatta
Pan troglodytes

platform_organism:
Macaca mulatta
Pan troglodytes

overall_design:
poly(A) enriched RNAs extracted from pooled samples of two brain regions: CBC and PFC of  chimpanzee and macaque, fragmented, revers transcribed to double-stranded cDNA using random hexamers. Sequencing libraries were prepared according to the paired-end non-strand-specific sample preparation protocol of Illumina. Each sample was sequenced in a separate lane in the Illumina Genome Analyzer II system, using the 75-bp paired-end sequencing protocol.
human data was downloaded from SRA [SRP005169]

first: Li

last: Zhongshan

first: Hindrike

last: Bammann

first: Zheng

last: Yan

first: Philipp

last: Khaitovich


title: H3K27me3 in human bladder transitional cell carcinoma cell line CL1207

city: Zurich

first: Valentina

last: Boeva

address: Universitatstrasse 6

department: Computer Science

country: Switzerland

institute: ETH Zurich

summary: The H3K27me3 ChIP-seq data for the human bladder transitional cell carcinoma cell line CL1207 were generated in order to detect regions of regional epigenetic silencing in this cell line and test the performance of several peak calling tools: CCAT (Xu et al., 2010) and HMCan (Ashoor et al., "HMCan – a tool to detect chromatin modifications in cancer samples using ChIP-seq data", submitted).

overall_design: The human bladder cancer cell line CL1207 was derived from a muscle-invasive bladder cancer (De Boer et al., 1997). 5x105 cells were immunoprecipitated per ChIP assay with 4 μg of rabbit polyclonal antibodies against trimethyl histone H3 lysine 27 (Upstate Biotechnology, Santa Cruz, CA) and  Dynabeads® Protein A (Invitrogen, Cergy Pontoise, France) in dilution buffer containing 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl at pH 8.0, and protease inhibitors. Six ChIP assays in the same experimental conditions were necessary to perform one ChIP-Seq experiment, so the total of 3x106 cells for each of the duplicates.

first: Aurélie

last: Hérault

first: François

last: Radvanyi


title: RNA sequencing of pheromone stimulated and unstimulated MATa and MATα Saccharomyces cerevisiae

city: Ithaca

first: Lori

last: Huberman

state: NY

address: 236 Tower Rd.

department: Plant Pathology and Plant-Microbe Biology, School of Integrative Plant Science

institute: Cornell University

summary: Haploid budding yeast has two mating types, defined by the alleles of the MAT locus, MATa and MATα. Mating occurs when two haploid cells of opposite mating types signal to each other using reciprocal pheromones and receptors, polarize and grow towards each other, and eventually fuse to form a single diploid cell.  The pheromones and receptors are necessary and sufficient to define a mating type, but other mating type-specific proteins make mating more efficient.  We examined the role of these proteins by genetically engineering “transvestite” cells that swap the pheromone, pheromone receptor, and pheromone processing factors of one mating type for another.  These cells can mate with each other, but their mating is inefficient.  By characterizing their mating defects and examining their transcriptomes, we found Afb1 (a-factor barrier), a novel MATα-specific protein that interferes with a-factor, the pheromone secreted by MATa cells.  We show that strong pheromone secretion is essential for efficient mating and that the weak mating of transvestites can be improved by boosting their pheromone production.  Using synthetic biology, it is possible to characterize the factors that control efficiency in biological processes.  In the case of budding yeast mating, selection for increased mating efficiency is likely to have continually boosted pheromone levels and the ability to discriminate between partners who make more (potentially fitter) and less (potentially less fit) pheromones. This sensitivity to which partner makes more pheromone comes at a cost: it means mating is not robust in situations where all potential partners make less pheromone.

overall_design: 4 conditions were analysed, each with 3 biological replicates.  The conditions were unstimulated MATa cells in YPD. Stimulated MATa cells in YPD+10nM α-factor.  Unstimulated MATα cells in YPD.  Stimulated MATα cells in YPD+10nM α-factor.

first: Andrew

middle: W

last: Murray


title: RNA-Seq and expression data from human adipose tissue

city: New York

first: Chenyi

last: Xue

institute: Columbia University


title: An epigenetic basis for lateral inhibition and lineage plasticity in intestinal differentiation

status: Public on Jan 06 2014

city: Boston

first: Fugen

last: Li

state: MA

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute


title: Analyses of the chromatin and transcriptional basis for lateral inhibition in isolated intestinal epithelial cells.

summary: We analyzed chromatin modifications, DNaseI-hypersensitive sites, and occupancy of a key secretory-lineage transcription factor, ATOH1. We found that lateral inhibition in the intestine occurs through ATOH1 exerting direct control within a broadly permissive chromatin state that is established in stem cells and is highly similar in specified progenitors of divergent potential.

overall_design: Mapping chromatin modifications (H3K4me2 and H3K27ac), DNaseI hypersensitivity (DHS), and ATOH1 binding sites in isolated intestinal crypt progenitors and mature intestinal villus cells.

first: Tae-Hee

last: Kim

first: Li-Lun

last: Ho

first: Annouck

last: Luyten

first: Kodandaramireddy

last: Nalapareddy

first: Henry

last: Long

first: Michael

last: Verzi

first: Ramesh

last: Shivdasani


title: MicroRNA profiling of rat hippocampus after status epilepticus induced by amygdala stimulation

city: Qingdao

first: Zhen

last: Sun

state: Shandong

address: No.5 Donghai Middle Road, Qingdao

institute: Qingdao Municipal Hospital, Taishan Medical University

summary: MicroRNAs (miRNAs) are small and endogenously expressed non-coding RNAs that negatively regulate the expression of protein-coding genes at the translational level. Emerging evidence suggests that miRNAs play critical roles in central nervous system under physiological and pathological conditions. However, their expression and functions in status epilepticus (SE) have not been well characterized thus far. Here, we characterized miRNA expression profile in rat hippocampus at 24 hours following SE induced by amygdala stimulation.

overall_design: Hippocampus miRNA profiles of experimental group and control group were generated by deep sequencing, using Hiseq 2000.

first: Jin-Tai

last: Yu


title: CLK:BMAL1 is a pioneer-like transcription factor [CLK_ChIP]

city: College Station

first: Jerome

last: Menet

state: TX

address: 301 Old Main Drive, Building 1530, ILSB

department: Dept of Biology

institute: Texas A&M University

summary: The mammalian circadian clock relies on the master genes CLOCK (CLK) and BMAL1 and drives rhythmic gene expression to regulate biological functions under circadian control. We recently uncovered a surprising disconnect between the rhythmic binding of CLK:BMAL1 on DNA and the transcription of its target genes, suggesting that they are regulated by as yet uncharacterized mechanisms. Here we show that rhythmic CLK:BMAL1 DNA binding promotes rhythmic chromatin opening. The underlying mechanisms include CLK:BMAL1 binding to nucleosomes and rhythmic chromatin modifications, including the incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLK:BMAL1, suggesting that the activity and the tissue-specific expression of these other transcription factors contribute to the genome-wide CLK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation, well beyond the current confines of developmental/cell specification.

overall_design: Mouse liver CLK ChIP-Seq signal on Mnase-digested or sonicated chromatin, at 2 different time point (ZT22 and ZT06) from the same mice. Libraries were sequenced using Ilumina HiSeq2000. For Mnase-digested chromatin, libraries contained a mononucleosome insert (e.g., ~147bp), whereas the insert was ~150-300bp for sonicated chromatin.

first: Stefan

last: Pescatore

last: Rosbash


title: CLK:BMAL1 is a pioneer-like transcription factor

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

overall_design: Refer to individual Series


title: Characterization of genome-wide transcriptional changes in liver and adipose tissues of ZDF (fa/fa) rats fed R-α-lipoic acid by next-generation sequencing

city: Lincoln

first: Jean-Jack

middle: M.

last: Riethoven

state: NE

address: 1901 Vine Street

department: Beadle Center for Biotechnology

institute: University of Nebraska-Lincoln

summary: We report on the characterization of lipogenic tissue transcriptional networks that support physiological responses of obese rats to a lipid-lowering bioactive food compound, R-a-lipoic acid (LA). Nine-week old male ZDF (fa/fa) rats were fed a chow diet supplemented with 3 g LA per kg diet or pair fed for two weeks. At the end of the trial, high-quality RNA was extracted from the liver and epididymal fat and subjected to transcriptome analysis using RNA-Seq technology. Results showed a substantially higher number of differentially expressed genes (DEG, q ≤ 0.05 and absolute log2(fold change) ≥ 1) in liver (110 genes) vs. epididymal fat (10 genes). Most epididymal fat DEG were also differentially expressed in liver and shared directionality of change. Gene Ontology (GO) analysis of these transcripts revealed significant enrichment of GO categories related to immune response, stress response, lipid metabolism, and carboxylic acid metabolic processes. Of interest interferon-related genes involved in defense against microorganism and innate immune response were induced by LA. Lipid metabolism-related transcript changes observed in LA-fed animals included downregulation of lipogenic genes (Pnpla3, Pnpla5, Elov6, Acly, Gpam, and Aacs) and concomitant upregulation of short-, medium- and long-chain fatty acid metabolic processes (Acot1, Acot2, Acsf2, and Crat). Transcriptional changes were accompanied by the lowering of abdominal adiposity and blood triacylglycerol levels. We conclude that LA dietary supplementation induces prominent gene expression changes in liver in support of significant improvement of whole-body lipid status

overall_design: For each tissue and feed combination (liver/LA+, liver/LA-, adipose/LA+, and adipose/LA-) the transcriptome was sequenced with 4 biological replicates.

first: Anjeza

last: Pashaj

first: Xiaohua

last: Yi

first: Mengna

last: Xia

first: Stephanie

last: Canny

first: Régis

last: Moreau


title: RNA methylation destabilizes developmental regulators in murine embryonic stem cells (m6A)

city: Cambridge

first: Yue

last: Li

state: Massachusetts

address: 32 Vassar Street, 32-D528

institute: Massachusetts Institute of Technology

summary: m6A-seq of mouse embryonic stem cell wildtype or mutant depleted of Mettl3 or Mettl14

overall_design: m6A-mRNA library for mouse embryonic stem cell each having one biological replicate were generated using HiSeq2000 v3 flowcell (Illumina) and sequenced for 100 bases with separate 7 base indexing read in a single lane.

first: Yang

last: Wang

first: Zhaolei

last: Zhang

first: Jing

middle: C

last: Zhao


title: Whole genome bisulfite-seq of two healthy males.

city: Houston

first: Govindarajan

last: Kunde Ramamoorthy

state: Texas

address: 1100 Bates Street

institute: Baylor College of Medicine

summary: Bisulfite-seq data sets were generated for peripheral blood lymphocyte (PBL) and hair follicle (HF) DNA from each of two healthy males.

overall_design: Examination of genome-wide CpG methylation two tissues (hair follicle and peripheral blood lymphocyte) from 2 healthy male individuals.

first: Robert

middle: A

last: Waterland


title: PGC7 and TET3 ChIP-seq results

status: Public on Jan 04 2014

city: Duarte

first: Xiaochun

address: 1500 E. Duarte Rd, Rm 202, Lippman-Graff Bldg

institute: city of hope

summary: PGC7 is a primordial germ cell (PGCs)-specific protein involved in epigenetic chromatin reprogramming in the zygote following fertilization. TET3 is a dioxygenase that catalyzes the conversion of the modified genomic base 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and plays a key role in epigenetic chromatin reprogramming in the zygote following fertilization.

overall_design: PGC7 participates in protection of DNA methylation in the maternal pronucleus by preventing conversion of 5mC to 5hmC by TET3 oxidization subsequent DNA demethylation. Thus we tested the co-localization of PGC7 and TET3 in specific loci by ChIP-seq assays.

first: Chunjing

last: Bian


title: Feeding with miR-184 affects the mRNA expression profile of queen larvae

city: Stanford

first: Xiangqian

last: Guo

address: 300 Pasteur Dr.

department: Department of Pathology

institute: Stanford University

summary: By comparing mRNA profiles of the two groups of bee larvae (treated by miR-184 and water), we found that there were 279 mRNAs which were differently expressed between treated and control larvae (ratio>2 and p<0.01), of which 200 mRNAs were down-regulated in treated larvae, and 79 mRNAs were down-regulated in controls.

overall_design: To further study the biological function and potential pathways of microRNAs with RNAi phenotypes, we measured mRNA profiles of bee larvae fed with miR-184 (treated) and water (control).

first: Songkun

last: Su

first: Geir

last: Skogerbø

first: Aung

last: Si

first: Shuanjin

last: Dai

first: Wenfeng

first: Zhiguo

first: Fang

last: Liu

first: Ruifeng

last: Ni

first: Yu

first: Shenglu

last: Chen

first: Shaowu

first: Runsheng


title: CLK:BMAL1 is a pioneer-like transcription factor [H2AZ-ChIP]

first: Jerome

last: Menet

overall_design: H2A.Z ChIP-Seq signal in the mouse liver over 6 time points of the 24h light:dark cycle, in wild-type and Bmal1-/- mice. Libraries containing a mononucleosome insert were sequenced using Ilumina HiSeq2000.

first: Stefan

last: Pescatore

first: Michael

last: Rosbash


title: In Vivo and In Vitro Dynamics of Undifferentiated Embryonic Cell Transcription Factor 1

status: Public on Jan 03 2014

city: Munich

middle: Johannes

last: Ziller

address: Kraepelinstrasse 2-10

department: Translational Psychiatry

country: Germany

institute: Max Planck Institute of Psychiatry

summary: Pluripotent stem cells retain the ability differentiate into the three germ layers and germ line. As a result, there is a major interest in characterizing regulators that establish and maintain pluripotency. The network of transcription factors continues to expand in complexity, and one factor, Undifferentiated embryonic cell Transcription Factor 1 (Utf1), has recently moved more into the limelight. To facilitate the study of Utf1, we report the generation and characterization of two reporter lines that enable efficient tracking, mapping and purification of endogenous Utf1. In particular, we include a built-in biotinylation system into our targeted locus that allows efficient and reliable pulldown. We also use this reporter to show the dynamic regulation of Utf1 in distinct stem cell conditions and demonstrate its utility for reprogramming studies. The multi-purpose design of the reporter lines enables many directions of future study and should lead to a better understanding of Utf1’s diverse roles.

overall_design: ChIP-Seq for endogenous and biotin tagged Utf1 in KH2 mouse embryonic stem cells

first: Alex

last: Meissner


title: Epigenetic priming of memory updating during reconsolidation to attenuate remote fear memories

first: Li-Huei

last: Tsai

department: Picower Institute for Learning and Memory

institute: MIT

summary: Remembrances of traumata range among the most enduring forms of memories. Despite the elevated lifetime prevalence of anxiety disorders, effective strategies to attenuate long-term traumatic memories are scarce. The most efficacious treatments to diminish recent (i.e., day-old) traumata capitalize on memory updating mechanisms during reconsolidation that are initiated upon memory recall. Here, we show that in mice successful reconsolidation-updating paradigms for recent memories fail to attenuate remote (i.e., month-old) ones. We find that whereas recent memory recall induces a limited period of hippocampal neuroplasticity mediated, in part, by S-nitrosylation of HDAC2 and histone acetylation, such plasticity is absent for remote memories. However, by using an HDAC2-targeting inhibitor (HDACi) during reconsolidation, even remote memories can be persistently attenuated. This intervention epigenetically primes the expression of neuroplasticity-related genes as revealed by whole genome RNA sequencing, which is accompanied by higher metabolic, synaptic and structural plasticity. Thus, applying HDACis during memory reconsolidation might constitute a treatment option for remote traumata.

overall_design: 3 biological replicates per group were analyzed. The material analyzed was whole hippocampi from one brain hemisphere, from which total RNA was extracted.

first: Gräff

last: Johannes

first: Tsai

last: Li-Huei


title: Long-range joining of intra-chromosomal DNA double-strand breaks

first: Frederick

last: Alt

address: 1 Blackfan Circle

department: Program in Cellular and Molecular Medicine

institute: Boston Children's Hospital

type: Genome variation profiling by high throughput sequencing

summary: Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100 to 200 kilobases (kb) apart within switch (S) regions in the immunoglobulin heavy chain locus (IgH).  CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sm and in a downstream acceptor S region, with a DSB in Sm being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells.  Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B cell-specific processes or by general aspects of chromosome architecture and DSB repair.  Previously, we found that B cells with two yeast I-SceI endonuclease targets in place of Sg1 undergo I-SceI-dependent class switching from IgM to IgG1 at 5-10% of normal levels.  Now, we report that B cells in which Sg1 is replaced with a 28 I-SceI target array, designed to increase I-SceI DSB frequency, undergo I-SceI-dependent class switching at almost normal levels.  High-throughput genome-wide translocation sequencing revealed that I-SceI-generated DSBs introduced in cis at Sm and Sg1 sites are joined together in T cells at levels similar to those of B cells.  Such high joining levels also occurred between I-SceI-generated DSBs within c-myc and I-SceI- or CRISPR/Cas9-generated DSBs 100 kb downstream within Pvt1 in B cells or fibroblasts, respectively. We suggest that CSR exploits a general propensity of intra-chromosomal DSBs separated by several hundred kb to be frequently joined together and discuss relevance of this finding for recurrent interstitial deletions in cancer.

overall_design: Comparison of frequency of long-range joining between I-SceI-induced DSBs at IgH and c-myc loci in different cell types by HTGTS

first: Monica

last: Gostissa

first: Bjoern

last: Schwer

first: Amelia

last: Chang

first: Junchao

last: Dong

first: Robin

middle: M

last: Meyers

first: Gregory

last: Marecki

first: Vivian

last: Choi

first: Roberto

last: Chiarle

first: Ali

last: Zarrin


title: Transcriptome-wide discovery of microRNA binding sites in human brain by Ago2 HITS-CLIP

city: Iowa City

first: Ryan

last: Boudreau

state: IA

address: 500 Newton Rd

institute: University of Iowa

first: Beverly

last: Davidson


title: Transcriptome-wide discovery of microRNA binding sites in human brain by Ago2 HITS-CLIP [Ago2-miRNA-target mRNA complexes]

summary: Here, seeking to gain insight into the array of transcripts engaged with miRNAs in human brain, we performed HITS-CLIP to profile transcriptome-wide Ago2:RNA interactions in a panel of eleven post-mortem adult human brain samples harvested from adult motor cortex and cingulate gyrus, regions associated with movement and psychiatric disorders .

overall_design:
High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP)
Eleven post-mortem adult human brain tissues were subjected to ultraviolet radiation to crosslink proteins with nucleic acids, and HITS-CLIP was performed as previously described by Chi et al Nature. 2009 Jul 23;460(7254):479-86 using an anti-Ago2 polyclonal antibody and some additional modifications.


title: Recipe for a busy bee: MicroRNAs in honey bee caste determination

first: Xiangqian

last: Guo

summary: Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food source, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7-215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4th to 6th day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and in morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee.

overall_design: We collected worker and royal jelly of the Italian honeybee (ZND No.1, Apis mellifera ligustica) at 73~90 hours (4th-day larvae), 97~114 hours (5th-day larvae), and 121~138 hours (6th-day larvae) after hatching. After total RNA was extracted  and quantified , relative equal amounts of total RNAs from each of the three sampling days were pooled into respectively worker and royal jelly samples, and the fraction of small RNAs less than 30nt long was retained and sequenced on the Illumina/Solexa high-throughput platform (HTP).

first: Songkun

last: Su

first: Geir

last: Skogerbø

first: Aung

last: Si

first: Shuanjin

last: Dai

first: Wenfeng

last: Li

first: Zhiguo

first: Fang

last: Liu

first: Ruifeng

last: Ni

first: Yu

first: Shenglu

last: Chen

first: Shaowu

last: Zhang

first: Runsheng


title: The Histone H3 Lysine 9 Methyltransferases G9a and GLP Regulate Polycomb Repressive Complex 2-Mediated Gene Silencing [RNA-Seq]

city: Paris

first: PONTIS

last: Julien

address: 35, rue Hélène BRION

country: France

institute: UMR7216 CNRS

summary: G9a/GLP and Polycomb Repressive Complex 2 (PRC2) are two major epigenetic silencing machineries, which in particular methylate histone H3 on lysines 9 and 27 (H3K9 and H3K27), respectively. Although evidence of a crosstalk between H3K9 and H3K27 methylations has started to emerge, their actual interplay remains elusive. Here, we show that PRC2 and G9a/GLP interact physically and functionally. Moreover, combining different genome-wide approaches, we demonstrate that Ezh2 and G9a/GLP share an important number of common genomic targets, encoding developmental and neuronal regulators. Furthermore, we show that G9a enzymatic activity modulates PRC2 genomic recruitment to a subset of its target genes. Taken together, our findings demonstrate an unanticipated interplay between two main histone lysine methylation mechanisms, which cooperate to maintain silencing of a subset of developmental genes.

overall_design: RNA-seq has been perform in triplicate on mES cell (TT2 : Wildtype, and KO G9a-/-)

first: Julien

last: Pontis

first: Chiara

last: Mozzetta

first: Slimane

last: Ait-Si-Ali


title: A miR-155-ruled microRNA hierarchy in dendritic cell maturation and macrophage activation

first: Anne

last: Dueck

state: Bavaria

address: Biedersteiner Str.29

department: Institute of Pharmacology and Toxicology

institute: TUM

summary: MicroRNAs (miRNAs) are small RNAs that play important regulatory roles in many cellular pathways. MiRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences on mRNAs and induce translational repression or mRNA decay. MiRNA expression can be controlled by transcription factors and can therefore be cell type- or tissue-specific. Here we have analyzed miRNA expression profiles in murine monocyte-derived dendritic cells (DCs) and macrophages upon stimulation with LPS, LDL, eLDL and oxLDL to identify not only stimuli-specific miRNA, but also to identify a hierarchical miRNA system involving miR-155. For this, miR-155 knockout dendritic cells and macrophages were also sequenced using the same stimuli.

overall_design: Sequencing of murine monocyte-derived dendritic cells and macrophages (each wild type and miR-155 knock out cells) matured and stimulated, respectively, by LPS, oxLDL, eLDL or LDL.

first: Alexander

last: Eichner

first: Michael

last: Sixt

first: Gunter

last: Meister


title: Three-dimensional modeling of the P. falciparum genome during the erythrocytic cycle reveals a strong connection between genome architecture and gene expression

city: Riverside

first: Karine

last: Le Roch

address: 900 University Ave

institute: University of California, Riverside

summary: The development of the human malaria parasite Plasmodium falciparum is controlled by coordinated changes in gene expression throughout its complex life cycle, but the corresponding regulatory mechanisms are incompletely understood. To study the relation between genome architecture and gene regulation in Plasmodium, we studied the genome structure of P. falciparum at three time points during its erythrocytic (asexual) cycle. Using chromosome conformation capture coupled with next-generation sequencing technology (Hi-C), we obtained high-resolution chromosomal contact maps, which we then used to construct a consensus three-dimensional genome structure for each time point. We observe strong clustering of centromeres, telomeres, ribosomal DNA and virulence genes, resulting in a complex architecture that cannot be explained by a simple volume exclusion model. Internal virulence gene clusters appear to be an important factor in shaping the genome architecture as they exhibit domain-like structures similar to topological domains in mammalian genomes. Midway during the cell cycle, at the highly transcriptionally active trophozoite stage, the genome adopts a more open chromatin structure with increased chromosomal intermingling. In addition, we observed reduced expression of genes located in spatial proximity to the repressive subtelomeric center, and colocalization of distinct groups of parasite-specific genes with coordinated expression profiles. Overall, our results are indicative of a strong association between the P. falciparum spatial genome organization and gene expression. Understanding the molecular processes involved in genome conformation dynamics could contribute to the discovery of novel antimalarial strategies.

overall_design: Analysis of the spatial organization of the P. falciparum genome at three stages of the erythrocytic cycle using chromosome conformation capture coupled with next generation sequencing (Hi-C). As a control, we included one sample for which chromatin contacts were not preserved by crosslinking of DNA and proteins.

first: Ferhat

last: Ay

first: Evelien

last: Bunnik

first: Nelle

last: Varoquaux

first: Sebastiaan

last: Bol

first: Jacques

last: Prudhomme

first: Jean-Philippe

last: Vert

first: William

last: Noble


title: The RON receptor tyrosine kinase promotes metastasis by triggering epigenetic reprogramming through the thymine glycosylase MBD4

status: Public on Jan 02 2014

city: Salt Lake City

first: Stephanie

last: Cunha

state: Utah

address: 2000 Circle of Hope

department: Oncological Science

institute: Huntsman Cancer Institute, University of Utah


title: CHD chromatin remodelers regulate separate gene sets and distinct stages of Dictyostelium development

city: Oxfors

first: James

last: Platt

address: Roosevelt Drive

institute: University of Oxford

summary: We utilized RNA-seq to profile expression levels in wild type AX2 cells and three mutants for the CHD family of ATP-dependent chromatin remodellers in Dictyostelium discoideum. Total RNA was polyA enriched and sequenced at two stages of development: in the growth stage (0H) and in 5H cAMP pulsed cells (5H). All mutants analysed were also in the AX2 background.

overall_design: Looking at mRNA levels in AX2, chdA-, chdB- and chdC-.


title: The RON receptor tyrosine kinase promotes metastasis by triggering epigenetic reprogramming through the thymine glycosylase MBD4 (RNA-Seq)

summary: Metastasis is the major cause of death in cancer patients, yet the genetic/epigenetic programs that drive metastasis are poorly understood. Here, we report a novel epigenetic reprogramming pathway that is required for breast cancer metastasis. Concerted differential DNA methylation is initiated by activation of the RON receptor tyrosine kinase by its ligand, macrophage stimulating protein (MSP). Through PI3K signaling, RON/MSP promotes expression of the G:T mismatch-specific thymine glycosylase MBD4. RON/MSP and MBD4-dependent aberrant DNA methylation results in misregulation of a specific set of genes. Knockdown of MBD4 reverses methylation at these specific loci, and blocks metastasis. We also show that the MBD4 glycosylase catalytic residue is required for RON/MSP-driven metastasis. Analysis of human breast cancers using a set of specific genes that are regulated by RON/MSP through MBD4-directed aberrant DNA methylation revealed that this epigenetic program is significantly associated with poor clinical outcome. Furthermore, inhibition of Ron kinase activity with a new pharmacological agent prevents activation of the RON/MBD4 pathway and blocks metastasis of patient-derived breast tumor grafts in vivo.

overall_design: Examination of 3 cell types.

first: Alana

last: Welm


title: Transcriptome-wide discovery of microRNA binding sites in human brain by Ago2 HITS-CLIP [Ago2-miRNA complexes]

first: Ryan

last: Boudreau

first: Beverly

last: Davidson


title: Genome-wide binding of TEAD4 in gastric cancer cell lines MKN28 and SNU216

city: Daejeon

first: Byungho

last: Lim

address: Medical Genomics Research Center, KRIBB, 125 Daehak-ro, Yuseong-gu

country: South Korea

institute: Korea Research Institute of Bioscience and Biotechnology (KRIBB)

summary: To access target binding sites of TEAD4 in gastric cancer, TEAD4 binding was investigated using MKN28 and SNU216 cell lines by ChIP-seq.

overall_design: MKN28 and SNU216 cell lines were cross-linked with formaldehyde, fractionated by sonication and immunoprecipitated by TEAD4 antibody. Immunoprecipitated DNA was prepared by generating libraries and sequenced by massive parallel sequencing.

first: Yong-Sung

last: Kim

first: Seon-Young


title: RNA methylation destabilizes developmental regulators in murine embryonic stem cells

first: Yue

last: Li

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

overall_design: Refer to individual Series


title: RNA-seq melanoma

city: Amsterdam

first: Rene

last: Bernards

address: Plesmanlaan 121

institute: Netherlands Cancer Institute

summary: Using a chromatin regulator-focused shRNA library, we found that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes  resistance to BRAF and MEK inhibitors. To investigate how SOX10 loss leads to drug resistance, we performed transcriptome sequencing (RNAseq) of both parental A375 (Ctrl. PLKO) and A375-SOX10KD (shSOX10-1, shSOX10-2) cells. To ask directly whether SOX10 is involved indrug resistance in BRAF(V600E) melanoma patients, we isolated RNA from paired biopsies from melanoma patients (pre- and post- treatment) , that had gained  BRAF or MEK inhibitor resistance . We performed RNAseq analysis to determine changes in transcriptome upon drug resistance.

overall_design: Investigate genes regulated by SOX10 and differntial gene expression between pre- and post-treatment biopsies. We use short hairpin RNA to suppression SOX10 in A375 cells and cells were harvested with trizol reagent for RNA isolation. For paired biopsies (patient samples) we collected the first biopsy before the initiation of treatment and the second biopsy after drug resistance developed. RNA was isolated from FFPE samples and subjected for RNA sequencing.

first: Chong

last: Sun

first: Iris

last: de Rink


title: The Histone H3 Lysine 9 Methyltransferases G9a and GLP Regulate Polycomb Repressive Complex 2-Mediated Gene Silencing

first: PONTIS

last: Julien


title: ChIP-seq anlysis of Sox2, Tfap2c, and Cdx2 in trophoblast stem cells.

city: Wako

first: Itoshi

last: NIKAIDO

state: Saitama

address: 2-1 Hirosawa

department: Center for Biosystems Dynamics Research

country: Japan

institute: RIKEN

summary: To understand the mechanism underlying the transcriptional regulation by Sox2, we analyzed genome-wide binding sites of Sox2, Tfap2c, and Cdx2 in trophoblast stem (TS) cells by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).

overall_design: ZHBTc4- and embryo-derived TS cell lines were maintained in the presence of FGF4 and mouse embryonic fibroblasts (MEFs).

first: Kenjiro

last: Adachi

last: Nikaido

first: Hiroki

last: Ura

last: Ueda

first: Hitoshi

last: Niwa


title: Nanog Independent Reprogramming to iPSCs with Canonical Factors

status: Public on Jan 01 2014

first: Brandi

middle: Nicole

last: Davis-Dusenbery

address: 7 Divinity Ave

department: SCRB

institute: Harvard University

summary: It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical reprogramming factors Oct4, Sox2, Klf4 and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog-/- fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPS cells from Nanog-/- fibroblasts that effectively contributed to chimeric mice.  Thus while Nanog may be an important mediator of reprogramming it is not required for establishing pluripotency in the mouse, even under standard conditions.  In order to further evaluate the equivalency of Nanog null iPSC to nanog null ESCs, we have performed RNAseq on two independent nanog null iPSC lines, as well as Nanog Null ESC, WT ESC and iPSCs as well as MEFs.  As a negativve control for reprogramming we have analyzed a partially reprogrammed iPSC line.

overall_design: 2-4 biological replicates each of 7 conditions (WT MEFs, WT ESC, WT iPSC, WT partially reprogrammed iPSC (piPS), Nanog null ESC, Nanog null iPSC clone G2 and Nanog null iPSC clone G5)

middle: N

first: Kevin

last: Eggan


title: Impact Of Age-Associated Increase In 2'-O-Methylation Of miRNAs On Aging And Neurodegeneration In Drosophila

city: Philadelphia

first: Ammar

last: Naqvi

state: Pennsylvania

address: 2716 South Street

department: Center for Data-Driven Discovery in Biomedicine

institute: Childrens Hospital of Philadelphia


title: A Whole Transcriptional Linkage Analysis of Gene Co-Regulation in Insecticide Resistant House Flies, Musca domestica

city: Auburn

first: Nannan

last: Liu

state: AL

address: 301 Funchess Hall

department: Entomology

institute: Auburn University

summary: Illumina HiSeq 2000 RNASeq was conducted on the mRNA fraction of three adult female Musca domestica strains. Two strains were the insecticide susceptible strains CS and aabys, which has five distinct recessive morphological markers, one on each of the five autosomes in M. domestica. The third strain was ALHF, which is a highly insecticide resistant strain obtained in Alabama.  The reference transcriptome was generated de novo using Trinity. The coding trimmed sequences from Augustus were then used as the mapping reference for the individual FASTQ files from each strain. trace reads were mapped using RSEM to generate FPKM estimates. Gene expression levels were compared to the ALHF strain using EdgeR to identify genes that were downregulated in both aabys and CS.

overall_design: RNA Seq profiling of the mRNA of adult Musca domestica with different autosomal contributions from insecticide resistant and susceptible strains (whole insect pooled samples)

first: William

last: Reid

first: Ming

first: Lee

last: Zhang


title: Transcription in Pronuclei and One- to Four-Cell Embryos Drives Early Development in a Nematode

city: Aurora

first: Jianbin

last: Wang

state: CO

address: 12801 East 17th Ave

department: Biochemistry and Molecular Genetics

institute: University of Colorado Denver

summary: A long-standing view of development is that transcription is silenced in the oocyte until early divisions in the embryo. The point at which major transcription is reactivated varies in different organisms, but is usually after the 2-cell stage. However, this model may not be universal. We used RNA-seq and exploited the protracted development of the parasitic nematode Ascaris suum, to provide a comprehensive time course of mRNA expression, degradation, and translation during early development. Surprisingly, we find that ~4,000 genes are transcribed prior to pronuclear fusion and in the 1-4 cell embryos. Intriguingly, we do not detect maternal contribution of many orthologs of maternal C. elegans mRNAs, but instead find these are newly transcribed in the A. suum zygote prior to pronuclear fusion. Ribosome profiling demonstrates that, in general, early embryonic mRNAs are not stored for subsequent translation, but are directly translated following their synthesis. The role of maternally contributed and zygotically transcribed genes differs between the nematodes A. suum and C. elegans despite the fact that the two nematodes appear to exhibit highly similar morphological patterns during early development. Our study indicates that major transcription can occur immediately after fertilization and prior to pronuclear fusion in metazoa, suggesting that newly transcribed genes appear to drive A. suum early development. Furthermore, the mechanisms used for controlling the timing of the expression of key conserved genes has been altered between the two nematodes, illustrating significant plasticity in the regulatory networks that play important roles in developmental outcomes in nematodes.

overall_design: A total of 28 samples are used for this study. The RNAs for the library construction are derived from 0.5 – 1 ml of synchronized embryo stages corresponding to a minimum of 0.5 billion embryos derived from > 1,000 female worms. The RNAs for polysome analysis are from pooled fractions of polysome gradient. For each sample, the amount of RNA for all polysome fractions is equivalent to the total RNA from the sample. Thus, the data from polysome profiling and RNA transcriptomes are considered as biological replicates. The level of the RNAs between each neighboring stages during development are considered as the reference for each other. Different sequencing strategies were applied for multiple libraries.

first: Julianne

last: Garrey

first: Richard

middle: E

last: Davis


title: ChIP-seq analysis of chromatin state plasticity in primary human adult erythroid progenitor cells

city: Memphis

first: Jian

last: Xu

state: Tennessee

address: 262 Danny Thomas Place, MS 345

department: Pathology

institute: St. Jude Children's Research Hospital

summary: We report a computational approach for investigation of chromatin state plasticity. We applied this approach to investigate an ENCODE ChIP-seq dataset profiling the genome-wide distribution of H3K27me3 in 19 human cell lines. We found that high plasticity regions (HPRs) can be divided into two functionally and mechanistically distinct groups, consisting of CpG island proximal and distal regions. We identified cell-type specific regulators correlating with H3K27me3 patterns at distal HPRs in ENCODE cell lines. Furthermore, we applied this approach to investigate mechanisms for poised enhancer establishment in primary human erythroid precursors. We predicted and validated a previously unrecognized role of TAL1 in modulating H3K27me3 patterns through interaction with additional cofactors, such as GFI1B. Our integrative approach provides mechanistic insights into chromatin state plasticity and is broadly applicable to other epigenetic marks.

overall_design: Analysis of genomic occupancy of H3K27me3, H3K27ac, GATA1, TAL1/SCL and GFI1B in primary adult human proerythroblasts by ChIP-seq.

first: Luca

last: Pinello

first: Stuart

middle: H

last: Orkin

first: Guo-Cheng

last: Yuan


title: Transcription factor Achaete-Scute homologue 2 initiates T follicular helper cell development

city: Bethesda

first: Lai

last: Wei

state: MD

address: 10 Center Dr. Room 2B47

institute: NIH/NEI/NCCAM


title: The Histone H3 Lysine 9 Methyltransferases G9a and GLP Regulate Polycomb Repressive Complex 2-Mediated Gene Silencing [ChIP-Seq]

first: PONTIS

last: Julien

overall_design: ChIP-seq has been performed for G9a and Ezh2 in wild type TT2 mES cells or in mES cells lacking both G9a and GLP (G9a-/-GLP-/-). As a control, input DNA was saved before immunoprecipitation. Note that the two inputs (Input_TT2_0 and Input_TT2_1) have been combined and used as control for Ezh2 and G9a ChIP-seq.

first: Julien

last: Pontis

first: Chiara

last: Mozzetta

first: Slimane

last: Ait-Si-Ali


title: Ago1 vs Ago2-IP small RNA deep-sequencing with age in Drosophila

first: Ammar

summary:
Drosophila miRNAs show distinct change in isoform distribution pattern with age.
Some miRNAs show accumulation of the short isoforms, while other miRNAs show the accumulation of the long isoforms with age.
The increase of the long isoforms of some miRNAs reflects increased 2'-O-methylated miRNA isoforms with age.
The increase in 2'-O-methylated miRNA isoforms reflected increased Ago2-loading, but not Ago1-loading of specific miRNA isoforms with age.
This raised a question on whether there is global shift in small RNA loading pattern between Ago1 and Ago2 with age.
To investigate change in small RNA loading pattern between Ago1 and Ago2 with age, we performed small RNA deep-sequencing of Ago1 vs Ago2-IP small RNAs at 3d and 30d in Drosophila.
This analysis revealed global increase of miRNA loading into Ago2, but not into Ago1 with age.

overall_design:
3d and 30d FLAG-HA-Ago2 male flies were collected.
Ago1 and Ago2 were immunoprecipitated by anti-Ago1 and anti-FLAG M2 beads respectively.
RNA was purified from the beads, P32-labeled, and small RNA fraction was gel-purififed.
Small RNA libraries were prepared using Illumina's TruSeq small RNA sample preparation kit (#RS-200-0012, Illumina, Inc. San Diego, CA), following the manufacturer's protocol.
The libraries were multiplexed and sequenced on HiSeq2000 platform (Illumina).

first: Masashi

last: Abe

first: Nancy

last: Bonini

first: Andrey

last: Grigoriev


title: Transcription factor Achaete-Scute homologue 2 initiates T follicular helper cell development [ChIP-Seq]

summary: In immune responses, activated T cells migrate to B cell follicles and develop to T follicular helper (Tfh) cells, a new subset of CD4+ T cells specialized in providing help to B lymphocytes in the induction of germinal centers 1-3. Although Bcl6 has been shown to be essential in Tfh cell function, it may not regulate the initial migration of T cells 4 or the induction of Tfh program as exampled by CXCR5 upregulation 5. Here, we show that the Achaete-Scute homologue 2 (Ascl2) gene that encodes a basic helix-loop-helix (bHLH) transcription factor 6, is selectively upregulated in its expression in Tfh cells. Ectopic expression of Ascl2 uniquely upregulates CXCR5 but not Bcl6 and downregulates CCR7 expression in T cells in vitro and accelerates T cell migration to the follicles and Tfh cell development in vivo. Combined transcriptome profiling and genome-wide occupancy analysis indicate that Ascl2 directly regulates Tfh-related genes while inhibits expression of Th1 and Th17 genes. Acute deletion of Ascl2 as well as blockade of its function with the Id3 protein in peripheral CD4+ T cells results in a failure in Tfh cell development and the germinal center response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances Tfh cell generation. Thus, Ascl2 critically and directly initiates Tfh cell development.

overall_design: Decide Ascl2 binding sites in CD4+ T cells

first: Xindong

last: Liu

first: Xiaowei

last: Yan

first: Chen

last: Dong


title: SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation

city: Seattle

first: Christopher

middle: Joel

last: Hale

state: WA

address: 1959 NE Pacific St., HSC H-458

institute: University of Washington

summary: RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana depends on the  synthesis of non-coding RNAs by nuclear RNA polymerase E (NRPE or Pol V) 1-3, but the mechanism by which Pol V is targeted is unknown. Here we show that genome-wide Pol V association with chromatin redundantly requires the  SU(VAR)3-9 homologs, SUVH2 and SUVH9. These proteins resemble histone  methyltransferases, however a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-Adenosyl methionine (SAM) binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins. SUVH2 and SUVH9 both contain SET- and RING-ASSOCIATED (SRA) domains capable of binding methylated DNA, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of the DNA METHYLTRANSFERASE 1, MET1, causes losses of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. By tethering SUVH2 with a zinc finger to an unmethylated epiallele of the homeodomain transcription factor FWA, we demonstrate that SUVH2 is sufficient to both recruit Pol V and establish DNA methylation and gene silencing. Our results suggest that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of the plant genome for epigenetic silencing.

overall_design: For wild type plants (ecotype Columbia) and suvh2 suvh9 double mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. For the small RNA sequencing nrpd1 and nrpe1 mutant plants were sequenced in parallel as controls. For the bisulfite sequencing data, the wildtype data and that of the suvh2 and suvh9 single mutants was previously published and is thus not submitted here. In addition, two replicates of whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of wildtype and suvh2 suvh9 mutant plants carrying a C-terminally epitope tagged (3XFLAG) NRPE1. Whole genome ChIP seq was also performed using a gifted endogenous NRPE1 antibody in a wildtype and met1 mutant background. Whole-genome bisulfite sequencing was also performed on fwa-4 epiallele plants transformed with the wild-type SUVH2 protein-coding construct containing a tethering zinc finger targeted to repeats at the fwa gene. For these transgenic libraries a line carrying a FLAG and fwa targeting zinc-finger tagged KRYPTONITE methyltransferase was bisulfite sequenced as a control (“FLAG-ZF-KYP”).


title: Non-canonical TSSs of TEs alter gene structure and expression in Drosophila melanogaster

city: Castanet

first: Matthias

last: Zytnicki

address: 24, Chemin de Borde-Rouge

department: Applied Mathematics and Informatics

institute: INRA

summary:
Background: Transposable elements (TEs) represent a substantial fraction of the genomes, playing a major role in evolution, as sources of genetic variability. To fully appraise the role of TE in the acquisition of genetic novelty in genome evolution, we must also consider the impact of their own transcriptional activity.
Results: We studied impact of TE transcriptional activity on gene using high-throughput RNA-Seq sequencing in Drosophila melanogaster. TEs, which turn out to be expressed in euchromatin as well as in heterochromatin, interact with genes at different levels. The observed transcription from TEs involve canonical or non-canonical transcription start sites (TSSs) distributed along their sequence. We also find evidences for potential bidirectional transcription from the TE promoter regions where the antisense transcript is co-opted by the host genome as TSSs of a gene. We found that active TEs seem to accumulate in the 5' upstream regions of the genes, and possibly provide an alternative transcript of the nearby gene. Indeed, predominantly, the TE transcript is collinear and overlapping the gene. Apart from the 5' upstream regions, we also found that most active TEs are transcribed on the gene transcript strand. Conversely, few transcripts from TE are anti sense with respect to the gene. This suggests that they have a disruptive action and are counter-selected. The only exceptions are for TEs located into introns, where they could provide another complex way of gene regulation, and in the 3' downstream region, where other mechanisms akin to siRNAs could take place. Finally, we noted several cases where the cryptic TSS is located on TE fragments corresponding to a low complexity sequence. Frequently these TE fragments appear to be over-represented when close to genes, suggesting a possible selected role.
Conclusion: Altogether, these results suggest that active transposable elements influence host gene transcription. It is likely that some features of transposable elements have been exaptated in order to enrich the genes repertoire by opening routes to sub- or neo-functionalization.

overall_design: Examination of the transcription produced by transposable elements in D. melanogaster


title: Hepatocyte Nuclear Factors 4a and 1a (Hnf4a and Hnf1a) Regulate Kidney Developmental Expression of Drug-Metabolizing Enzymes and Drug Transporters

city: La Jolla

first: Gleb

last: Martovetsky

state: CA

address: 9500 Gilman Drive

institute: UCSD

summary: The transcriptional regulation of drug-metabolizing enzymes and transporters (here collectively referred to as DMEs) in the developing proximal tubule is not well understood. As in the liver, DME regulation in the PT may be mediated through nuclear receptors which are thought to “sense” deviations from homeostasis by being activated by ligands, some of which are handled by DMEs, including drug transporters. Systems analysis of transcriptomic data during kidney development predicted a set of upstream transcription factors, including Hnf4a and Hnf1a, as well as Nr3c1 (Gr), Nfe2l2 (Nrf2), Ppara, and Tp53. Motif analysis of cis-regulatory further suggested that Hnf4a and Hnf1a are the main transcriptional regulators in the PT. Available expression data from tissue-specific Hnf4a KO tissues revealed that distinct subsets of DMEs were regulated by Hnf4a in a tissue-specific manner. ChIP-seq was performed to characterize the PT-specific binding sites of Hnf4a in rat kidneys at three developmental stages (prenatal, immature, adult), which further supported a major role for Hnf4a in regulating PT gene expression, including DMEs. In ex vivo kidney organ culture, an antagonist of Hnf4a (but not a similar inactive compound) led to predicted changes in DME expression, including among others Fmo1, Cyp2d2, Cyp2d4, Nqo2, as well as organic cation transporters and organic anion transporters Slc22a1(Oct1), Slc22a2 (Oct2), Slc22a6 (Oat1), Slc22a8(Oat3), and Slc47a1(Mate1). Conversely,  overexpression of Hnf1a and Hnf4a in primary mouse embryonic fibroblasts (MEFs), sometimes considered a surrogate for mesenchymal stem cells, induced expression of several of these proximal tubule DMEs, as well as epithelial markers and a PT-specific brush border marker Ggt1. These cells had organic anion transporter function. Taken together, the data strongly supports a critical role for HNF4a and Hnf1a in the tissue-specific regulation of drug handling and differentiation toward a PT cellular identity.

overall_design: Hnf4a binding was examined in rat kidneys at three timepoints (E20, P13 and Adult) and p300 binding was examined in adult rat kidney cortex tissue using ChIP-seq. Four corresponding input DNA samples were used as controls for peak calling.

first: James

last: Tee

first: Sanjay

middle: K

last: Nigam


title: Tolerance to acetic acid is improved by mutations of the TATA-binding protein gene

city: Seoul

first: In-Geol

last: Choi

address: Anam-dong Seongbuk-gu

department: Biotechnology

institute: Korea University

summary: To identify genes responsible for the enhanced tolerance, the transcriptome profile of one acetic acid-tolerant strain was compared with that of a control strain.

overall_design: RNAseq analysis of acetic acid-tolerant strain and control strain.

first: Wankee

last: Kim

first: Wonja

first: Kyoung


title: HOT regions are CpG dense promoters in C. elegans and humans

city: Cambridge

first: Przemyslaw

middle: Aleksander

last: Stempor

state: United Kingdom

address: Tennis Court Road

department: The Gurdon Institute

institute: University of Cambridge

summary: ChIP seq of Cfp-1 and H3K4me3 in C. elegans late embryos

overall_design: The coding region of F52B11.1a (cfp-1) was PCR amplified from N2 genomic DNA using Phusion polymerase (Finnzymes) and Gateway cloned into pDONR221.   The cfp-1 coding region was then recombined into the MosSCI compatible vector pCFJ201 (which targets Mos site Mos1(cxTi10882) chrIV ) downstream of the dpy-30 promoter and upstream of gfp::tbb-2 3’UTR (Zeiser et al. 2011) to generae strain JA1597 expressing GFP tagged Cfp-1 protein.  Late embryos were obtained by aging embryos collected by hypochlorite treatment 3.5 hrs prior to flash freezing in liquid nitrogen.  Formaldehyde-fixed chromatin extracts and chromatin immunoprecipitations were as in (Kolasinska-Zwierz et al. 2009) except that DNA was sonicated to a size range of 200-400bp.  ChIP assays were performed in 1 ml extract (1 mg protein) in FA buffer with 10 micrograms of anti-GFP rabbit serum (Abcam ab290) and anti-H3K4me3 (Abcam ab8580) individually.  DNA sequencing libraries were constructed according using the Illumina Truseq sequencing kit and were sequenced on the Illumina platform.

first: Julie

last: Ahringer


title: ChIP-seq investigation of H-NS of Escherichia coli grown in the presence and absence of an antibiotic "Bicyclomycin", a potential inhibitior of transcription termination factor rho.

city: Bangalore

first: Deepti

last: Chandraprakash

state: Karnataka

address: GKVK, bellary road

institute: National Centre for Biological Sciences

summary: We report that inhibition of rho by treatment with bicyclomycin, a potential inhibitor of rho affects H-NS binding to chromosome across most of the binding sites. For the current study, we have flag tagged H-NS by homologous recomination method. Cells were grown in the presence and absence of antibiotic bicylomyin. Chromatin immunoprecipitation was performed and anti flag antibody was used for immunoprecipitation. Immunoprecipitated samples both from BCM- and BCM + conditions were sequenced using HiSeq 1000 model.  Input DNA was used as control for this experiment which was also sequenced. The 50-mer reads obtained were then mapped to Escherichia coli Mg1655 K-12 genome. Further analysis was carried out to calculate the ChIP signal score. We observed a drop in ChIP signal for bcm+ condition as compared to bcm-.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: ChIP-seq data generated by Hiseq 1000 model of samples with bcm+ and bcm- in biological duplicates along with Input DNA as control


title: small RNA deep-sequencing with oxidation/β-elimination with age in Drosophila

first: Ammar

middle: S

last: Naqvi

summary:
Drosophila miRNAs show distinct change in isoform distribution pattern with age.
Some miRNAs show accumulation of the short isoforms, while other miRNAs show the accumulation of the long isoforms with age.
The increase of the long isoforms of some miRNAs reflects increased 2'-O-methylated miRNA isoforms with age.
This raised a question on whether additional miRNAs show increased 2'-O-methylated miRNA isoforms with age.
To investigate change in 2'-O-methylated small RNA population with age, we performed small RNA deep-sequencing of oxidized or non-oxidized small RNAs at 3d and 30d in Drosophila.
This analysis revealed change in global pattern of 2'-O-methylated miRNA isoforms with age.

overall_design:
3d and 30d wild-type male flies were collected.
Total RNA was prepared with Trizol reagent, followed by oxidation/β-elimination assay or the control treatment (no-oxidation).
Small RNA libraries were prepared using Illumina's TruSeq small RNA sample preparation kit (#RS-200-0012, Illumina, Inc. San Diego, CA), following the manufacturer's protocol.
The libraries were multiplexed and sequenced on HiSeq2000 platform (Illumina).

first: Masashi

first: Nancy


title: The Arabidopsis Zinc Finger Protein 3 integrates ABA and light signaling in seed germination and plant development

city: Morahalom

first: Balazs

last: Horvath

address: Vallalkozok u. 7.

department: NGSP

country: Hungary

institute: SeqOmics Biotechnology Ltd.

summary: ZFP3, a nuclear C2H2 zinc finger protein acts as a negative regulator of ABA- suppressed germination. Regulated over-expression of ZFP3 and closely related ZFP1, ZFP4, ZFP6 and ZFP7 confers ABA insensitivity to seed germination while the zfp3,zfp4 double mutant displays enhanced ABA susceptibility. Reduced expression of a large set of ABA-induced genes such as RAB18 and transcription factors ABI3, ABI4, ABI5 and RGL2 in ZFP3ox seedling suggests that ZFP3 indeed negatively regulates ABA signaling. Analysis of ZFP3ox plants revealed multiple phenotypic alterations such as semidwarf growth habit, defects in fertility and enhanced sensitivity of hypocotyl elongation to red but not to far-red or blue light. Analysis of genetic interactions with phytochrome and abi mutants suggested that ZFP3 amplifies red light signals perceived by photoreceptors other than phyB, and controlled by ABI5 downstream of ZFP3.

overall_design: Comparison of ZFP3 overexpressing and wild type Arabidopsis seedlings

first: Mary

middle: P

last: Joseph

first: László

last: Szabados

first: István

last: Nagy

first: Balázs

last: Horváth


title: Study of poplar shoot apex methylome response to variations in soil water avaialbility

city: Uppsala

first: Clement

last: Lafon-Placette

address: Almas Allé 5

institute: SLU

type: Methylation profiling by genome tiling array

summary: In a context of climate changes, water availability is expected to become a limiting factor for plant growth and to have an impact on forest health. In order to identify genes involved in shoot phenotypic plasticity in response to variations in water availability in trees, DNA methylation patterns were investigated in the shoot apical meristem (SAM) of Populus deltoides × P. nigra hybrid cuttings submitted to a moderate water deficit followed by a rewatering step. Transcriptomic response was also studied and is another GEO submission.

overall_design: Populus deltoides × P. nigra 'Carpaccio' hybrid cuttings were submitted to 3 water conditions: non-limiting, water deficit and water deficit/rewatering cycle (14 days treatment). At the end of the experiment, buds were collected and SAM maually isolated for 6 individuals per condition. DNA was extracted from 3 individuals per condition and pooled. For each condition, methylated DNA was isolated using a MethylDNA Immunoprecipitation (MeDIP) and both total DNA and MeDIP fraction were hybridised to microarrays.

first: Clément

first: Alain

last: Delaunay

first: Irène

last: Hummel

first: Béline

last: Jesson

first: Marie-Béatrice

last: Bogeat-Triboulot

first: Franck

last: Brignolas

first: Stéphane

last: Maury


title: Expression analysis of Candida albicans following a block in mitotic exit

city: Exeter

first: Steven

last: Bates

address: GP Building, Stocker Road

department: Biosciences

institute: University of Exeter

summary: Candida albicans demonstrates three main growth morphologies yeast, pseudohyphal and true hyphal forms.  Cell separation is distinct in these morphological forms and the process of separation is closely linked to the completion of mitosis and cytokinesis.  In Saccharomyces cerevisiae the small GTPase Tem1 is known to initiate the mitotic exit network, a signalling pathway involved in signalling the end of mitosis and initiating cytokinesis and cell separation.  We have characterised the role of Tem1 in C. albicans, and demonstrate that it is essential for mitotic exit and cytokinesis, and that this essential function is signalled through the kinase Cdc15.  Consistent with its role in activating the mitotic exit network Tem1 localised to spindle pole bodies in a cell cycle dependent manner.  Cells depleted of Tem1 displayed highly polarised growth but ultimately fail to complete cytokinesis and re-enter the cell cycle following nuclear division.  At the transcriptional level genes downregulated following the depletion of Tem1 where significantly enriched for genes whose expression peaks early in the cell cycle and for those associated with glycolysis.

overall_design: For expression analysis triplicate RNA samples were generated from exponentially growing C. albicans yeast cells and from cells following 8 h inhibition of TEM1 expression.  Gene expression was determined using RNA-seq on the Illumina HiSeq 2000 platform.


title: Four distinct types of dehydration stress memory genes in Arabidopsis thaliana

city: Lincoln

first: Jean-Jack

last: Riethoven

address: 1901 Vine Street

department: Beadle Center for Biotechnology

institute: University of Nebraska-Lincoln

summary: Arabidopsis plants that have experienced stress from water withdrawal show an improved ability to tolerate subsequent exposures as a ‘memory’ from the previous stress. This physiological stress memory is associated with ‘transcriptional memory’ illustrated by a subset of dehydrations stress responding genes that produce significantly different transcript amounts during repeated dehydration stresses relative to their response in the first. Here we report the genome-wide representation of dehydration stress transcriptional memory genes in A. thaliana. We identify four novel transcription patterns in response to repeated dehydration stress treatments. The nature of the proteins encoded by genes from each type of memory-response pattern is analyzed and the consequences of the genes’ memory behavior are considered in the context of possible biological relevance. The memory behavior of genes co-regulated by the dehydration/ABA and other abiotic stress and hormone responding pathways suggested that the crosstalk at the transcriptional level between them was affected as well. The intensity and the nature of specific biochemical, membrane, chloroplast, and stress response-related interactions during multiple exposures to dehydration stress are different from the responses to a single dehydration stress. The results reveal additional, hitherto unknown, levels of complexity of the plants’ transcriptional behavior when adjusting and adapting to recurring water deficits.

overall_design: For each condition (water, S1, and S3) the transcriptome was sequenced for two replicates. The watered condition is considered the control.

first: Yong

last: Ding

first: Ning

last: Liu

first: Laetitia

last: Virlouvet

first: Michael

last: Fromm

first: Zoya

last: Avramova