title: Embryonic attenuated Wnt/β-catenin signaling defines niche location and long-term stem cell fate in hair follicle

city: Beijing

first: huanwei

last: huang

address: 7 Science Park Rd., ZGC life science park

institute: NIBS

summary: Long-term adult stem cells sustain tissue homeostasis and regeneration throughout the lifetime of an organism. They are thought to originate from embryonic progenitors that acquired long-term self-renewal ability and multipotency at the end of organogenesis. We aim to mechanistically understand the embryonic origin of localized hair follicle stem cells. Here we discover embryonic progenitor cells occupying upper hair peg location; expressing a stem cell marker Nfatc1, contribute to long-term hair follicle stem cells, while the ones locating in the lower hair peg, expressing Shh, acquire differentiated cell fate. To understand the underlying mechanism leading to niche-induced HFSC specification, we profiled the expression of mRNA isolated from purified hair peg cells expressing Shh or Nfatc1. Based on DAVID functional gene annotation analysis, among genes that were expressed 2 fold more in Shh+ cells than in Nfatc1+ cells, we found that genes of the Wnt signaling pathway were enriched prominently. Base on these result and further genetic study, we suggest when hair placode develops to hair peg, a localized Wnt/β-catenin signal free zone emerges. Embryonic progenitors residing in this region gain expression of stem cell markers compared to their precursors and become definitive long-term hair follicle stem cells.

overall_design: Shh or Nfatc1 expressing cells at hair peg stage were labelled by genetic tools. Then mRNA isolated from purified hair peg cells expressing Shh or Nfatc1 were profiled.

first: Zijian

last: Xu

first: Huanwei

last: Huang

first: Ting


title: Advancing Trypanosoma brucei genome annotation through ribosome profiling and spliced leader mapping

status: Public on Dec 22 2015

city: SEATTLE

first: Gowthaman

last: Ramasamy

state: WASHINGTON

address: 307 WESTLAKE AVE N

department: GHBC

country: USA

institute: SEATTLE BIOMEDICAL RESEARCH INSTITUTE

type: Expression profiling by high throughput sequencing

summary: Since the initial publication of the trypanosomatid genomes, curation has been ongoing. Here we apply the technique of ribosome profiling to Trypanosoma brucei, identifying 223 new coding regions by virtue of ribosome occupancy in the corresponding transcripts. A small number of these putative genes correspond to extra copies of previously annotated genes but 85% are novel. The median size of these novels CDSs is small (74 aa) indicating that past annotation work has excelled at detecting large CDSs. Of the unique CDSs discovered here, over half have candidate orthologues in other trypanosomatid genomes, most of which were not yet annotated as genes. Still, approximately one-third of the new CDSs were found only in T. brucei subspecies. When combined with RNA-seq and spliced leader mapping, we were able to definitively revise the start sites for 430 CDSs as compared to the current gene models. Such data also allowed us to use a structured approach to eliminate 701 putative genes as protein-coding. Finally, the data pointed to several regions of the genome that had sequence errors that altered coding region boundaries.

overall_design: Ribosome profiling and mRNA libraries were constructed in triplicate from in vitro  PCF and in vivo  BF lifestages of theT. brucei Treu927 and in vitro T. brucei  Lister427, to evaluate role of translational gene regulation

first: Marilyn

last: Parsons

first: Bryan

middle: C

last: Jensen

first: Elton

middle: J

last: Vasconcelos

first: Peter

last: Myler


title: A microfluidic platform enabling single cell RNA-seq of multigenerational lineages

city: Cambridge

first: Robert

last: Kimmerling

state: Massachusetts

department: Biological Engineering

institute: MIT

summary: We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multigenerationa lineage tracking under controlled culture conditions.

overall_design: Examination of lineage and cell cycle dependent transcriptional profiles in two cell types

first: Scott

middle: R

last: Manalis

first: Alex

middle: K

last: Shalek


title: Proteogenomics analysis reveals specific genomic orientations at distal regulatory regions composed by non-canonical histone variants

city: Philadelphia

first: Kyoung Jae

last: Won

address: 3400 Civic Center Blvd

department: Genetics

institute: University of Pennsylvania

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Histone variants (H3.1, H3.3, H2A, and macroH2A) were studied about their proteomic and genomic distribution in Hela cells

overall_design:
HeLa S3 cells stably expressing either FLAG-tagged H3.1, and H3.3 were grown in suspension in Joklik media containing 10% newborn calf serum (Hyclone), 1% GlutaMAX (Invitrogen), and 1% penicillin-streptomycin, and cells were harvested at log phase.
Nuclei were isolated, mononucleosomes were subsequently obtained from these cell lines.  Cells were lysed in hypotonic TMSD buffer to isolate nuclei, which were then digested with micrococcal nuclease. The resulting mononucleosomes were immunoprecipitated with anti-FLAG M2-agarose beads (Sigma) and eluted with FLAG peptide (Sigma)

first: Benjamin

last: Garcia

first: Inchan

last: Choi

first: Kyoung-Jae


title: Genome-wide maps of KDM5 binding in drosophila adults

city: Bronx

first: Julie

last: Secombe

state: United states

address: 1300 Morris park ave. Ullmann 809

institute: Albert einstein college of medicne

summary: To determine which genes affected by loss of KDM5 in adults were direct targets, we carried out KDM5 ChIP-seq analyses. To valide  this data, we utilized a previously generated fly strain in which the sole source of KDM5 is from a transgene expressing an HA tagged form of KDM5 expressed under the control of its endogenous promoter. Comparing genome-wide gene expression and KDM5  binding analyses in Drosophila adults, we demonstrate the primary function of KDM5 in adults is to activate gene expression KDM5. To investigate the link between KDM5 and H3K4me3, we carried out anti-H3K4me3 ChIP-seq from wildtype adults . Genome-wide, KDM5 and H3K4me3 peaks showed a similar distribution, with both peaking at the transcription start site (TSS) showed a striking overlap with the presence of H3K4me3.

overall_design: Examination of KDM5 binding and histone H3K4me3 modifications in drosophila adults

first: Xingyin

last: Liu


title: TNF and CD28 signaling play unique but complementary roles in the systemic recruitment of innate immune cells after Staphylococcus aureus enterotoxin A inhalation

city: Farmington

first: Julia

last: Svedova

state: CT

address: 263 Farmington Ave

department: Immunology

institute: UConn Health

summary: Staphylococcus aureus enterotoxins cause debilitating systemic inflammatory responses, but how they spread systemically and trigger cascading inflammation is unclear. Here, we showed in mice that after inhalation, Staphylococcus aureus enterotoxin A rapidly entered the bloodstream and induced T cells to orchestrate systemic recruitment of inflammatory monocytes and neutrophils. To study the mechanism used by specific T cells that mediate this process, a systems approach revealed inducible and non-inducible pathways as potential targets. It was found that TNF induced neutrophil entry into the peripheral blood, while CD28 signaling, but not TNF, was needed for chemotaxis of inflammatory monocytes into blood and lymphoid tissue. However, both pathways triggered local recruitment of neutrophils into lymph nodes. Thus, our findings revealed a dual mechanism of monocyte and neutrophil recruitment by T cells relying on overlapping and non-overlapping roles for the non-inducible costimulatory receptor CD28 and the inflammatory cytokine TNF. During sepsis, there might be clinical value in inhibiting CD28 signaling to decrease T cell-mediated inflammation and recruitment of innate cells while retaining bioactive TNF to foster neutrophil circulation.

overall_design: The purpose of this analysis was to determine changes in gene expression in SEA-specific Vβ3+ T cells and bystander T Vβ14+ cells 40 min after SEA or vehicle inhalation.The samples were collected from three independent experiments with total n=3 per group. Three groups of samples were prepared: vehicle Vβ3+ T cells, SEA Vβ3+ T cells, and SEA Vβ14+ T cells.

first: Anthony

middle: T

last: Vella


title: The Analysis of Genes that Influence Sheep Follicular Development by Different Nutrition Levels during the Luteal Phase using Expression Profiling

city: Nanjing

first: Feifei

last: Luo

state: Jiangsu Province

address: #1 Weigang

department: College of Animal Science & Technology

country: China

institute: Nanjing Agricultural University

summary:
Purpose: Nutrition is an important factor that regulates reproductive performance of sheep and affects follicle development. However, the correlation between nutrition and follicle development is poorly understood at molecular level. In order to study its possible molecular mechanisms, we performed expression profiling of granulosa cells isolated from sheep that were fed with different nutrition levels during luteal phase.
Methods: To do this, ewes received a maintenance diet (M) and their estrus was synchronized by intravaginal progestogen sponges for 12 days. Ewes were randomly divided into the short-term dietary restricted group (R; 0.5×M) and nutrient-supplemented group (S; 1.5×M). RNA samples were extracted from granulosa cells. Transcriptome libraries from each group were constructed by Illumina Sequencing.
Results: In 18,468 detected genes, 170 genes were significantly differentially expressed; of which, 140 genes were up-regulated and 30 genes were down-regulated in group S relative to group R. These genes could be candidates regulating follicular development in sheep. GO, KEGG and Clustering analyses were performed.
Conclusions: These data in our study are abundant genomic recourse to expand our understanding of the molecular and cellular events underlying follicle development.

overall_design: Sixteen multiparous Hu sheep (3-4 years; 42.2 ± 1.2 kg) were randomly assigned to two groups: the short-term nutrient-supplemented group (S; n=8), and the dietary restricted group (R; n=8).Expression profiling of granulosa cells were performed by Illumina sequencing platform (HiSeq 2000).

first: Ruoxin

last: Jia

first: Shijia

last: Ying

first: Ziyu

last: Wang

first: David

last: Doe


title: Loss of MEN1 activates DNMT1 implicating DNA hypermethylation as a driver of MEN1 tumorigenesis

first: Steven

last: Libutti

state: NY

address: 1300 Morris Park Ave

department: Surgery

institute: Albert Einstein College of Medicine

type: Methylation profiling by high throughput sequencing

summary: Multiple endocrine neoplasia type 1 (MEN1) syndrome is the result of mutations in the MEN1 gene and results in tumor formation via mechanisms that are not well understood. Using a novel genome-wide methylation analysis, we studied tissues from patients with MEN1-parathyroid tumors, tissues from Men1 knockout (KO) mouse models, and Men1 null mouse embryonic fibroblast (MEF) cell lines. We demonstrated that the inactivation of menin (the protein product of MEN1) results in increased activity of DNA (cytosine-5)-methyltransferase 1 (DNMT1) by retinoblastoma-binding protein 5 (Rbbp5) activation in MEN1 tumor tissues. The increased activity of DNMT1 mediated global DNA hypermethylation, which in turn resulted in aberrant activation of the Wnt/β-catenin signaling pathway through inactivation of Sox regulatory genes. Our study provides important insights into the possible regulatory role of menin in DNA methylation and its impact on the pathogenesis of MEN1 tumor development.

overall_design: Global DNA methylation in tissues from patients with MEN1-parathyroid tumors. Thirty-eight human parathyroid specimens were used: 13 sporadic (non-MEN1) parathyroid adenomas, 12 MEN1-parathyroid tumors, 4 parathyroid carcinomas, and 9 normal parathyroids.

first: Ziqian

last: Yuan

first: Carmen

last: Sanchez Claros

first: Masako

last: Suzuki

first: Elaine

last: Maggi

first: Justin

middle: D

last: Kaner

first: Noah

last: Kinstlinger

first: Thomas

last: Quinn

first: Rula

last: Geha

first: Amanda

last: Corn

first: Jessica

last: Pastoriza

first: Qiang

last: Jing

first: Asha

last: Adem

first: Hao

last: Wu

first: Girum

last: Alemu

first: Deyou

middle: M

last: Zheng

first: John

last: Greally


title: Next generation sequencing of miRNAs in folic acid-induced kidney damage

city: Boston

first: Kathryn

last: Pellegrini

address: 77 Avenue Louis Pasteur

department: Renal Division, Department of Medicine

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

type: Non-coding RNA profiling by high throughput sequencing

summary: We have used small RNA sequencing to measure the expression of all miRNAs present in the kidneys during the development of folic acid-induced kidney injury and fibrosis. From this miRNA expression profile, we identified miRNAs associated with different stages and aspects of kidney damage.

overall_design: BALB/c mice were injected with 250mg/kg of folic acid and kidneys were collected at 0, 1, 2, 3, 7, 14, and 28 days. Total RNA was prepared for sequencing using the Illumina TruSeq Small RNA Sample Preparation Kit, and analyzed on an Illumina HiSeq 2000.

middle: L

first: Vishal

middle: S

last: Vaidya


title: Exogenous rescue of KLHL9 in SF210 cell line

city: New York

first: James

last: Chen

state: NEW YORK

address: 1130 St Nicholas Ave

department: Systems Biology

institute: Columbia University

summary: Rescue of KLHL9 (WT) expression in KLHL9 null cell line, SF210, causes significant reprogramming away from a mesenchymal lineage.

overall_design: An inducible expression vector carrying KLHL9 or GFP was stably introduced into SF210 cells via lentivirus transduction. Individual lines were isolated via clonal dilution and induced using doxycycline.


title: Truncation of LOC100288798 (SLC38A4-AS) lncRNA in human haploid KBM7 cell line

city: Vienna

first: Aleksandra E

last: Kornienko

address: Lazarettgasse 14, AKH BT 25.3, 3rd floor

country: Austria

institute: CeMM  Research Center for Molecular Medicine of the Austrian Academy of Sciences

summary: Many thousand long non-coding (lnc) RNAs are mapped in the human genome. Time consuming studies using reverse genetic approaches by post-transcriptional knock-down or genetic modification of the locus demonstrated diverse biological functions for a few of these transcripts. The Human Gene Trap Mutant Collection in haploid KBM7 cells is a ready-to-use tool for studying protein-coding gene function. As lncRNAs show remarkable differences in RNA biology compared to protein-coding genes, it is unclear if this gene trap collection is useful for functional analysis of lncRNAs. Here we use the uncharacterized LOC100288798 lncRNA as a model to answer this question. Using public RNA-seq data we show that LOC100288798 is ubiquitously expressed, but inefficiently spliced. The minor spliced LOC100288798 isoforms are exported to the cytoplasm, whereas the major unspliced isoform is nuclear localized. This shows that LOC100288798 RNA biology differs markedly from typical mRNAs. De novo assembly from RNA-seq data suggests that LOC100288798 extends 289kb beyond its annotated 3' end and overlaps the downstream SLC38A4 gene. Three cell lines with independent gene trap insertions in LOC100288798 were available from the KBM7 gene trap collection. RT-qPCR and RNA-seq confirmed successful lncRNA truncation and its extended length. Expression analysis from RNA-seq data shows significant deregulation of 41 protein-coding genes upon LOC100288798 truncation. Our data shows that gene trap collections in human haploid cell lines are useful tools to study lncRNAs, and identifies the previously uncharacterized LOC100288798 as a potential gene regulator.

overall_design: We cultured and processed 8 KBM7 cell lines in one batch. These cell lines were: two wild type KBM7 cells (WT2 and WT3), two monoclonal KBM7 cell lines with gene trap cassette insertions outside of the body of LOC100288798 (C1 and C2), two independently obtained KBM7 clones with gene trap cassette insertion 3kb downstream LOC100288798 transcriptional start site (TSS) (3kb1 and 3kb2), one independently obtained KBM7 clone with gene trap cassette insertion 100kb downstream LOC100288798 TSS replicated twice at the thawing step (100kb1 and 100kb2). We isolated total RNA from all th 8 cell lines, applied DNAseI treatment and ribosomal RNA depletion, and thhen prepared strand-specific RNA-seq libraries, which were pooled in equal molarities and sequenced using Illumina HiSeq 2000 (8 pooled samples were sequence on 2 lanes). We performed 50bp single-end RNA-seq. We used these 8 samples (4 untreated: WT2, WT3, C1, C2 and 4 treated:3kb1, 3kb2, 100kbk1, 100kb2) to analyze genome-wide gene deregulation associated with LOC100288798 lncRNA truncation

first: Aleksandra

middle: E

first: Florian

last: Pauler

first: Irena

last: Vlatkovic


title: Early and late response of Nematostella vectensis transcriptome to heavy metals

city: HAIFA

first: Tamar

last: Lotan

address: 199 Abba Khoushy Ave.   Mount Carmel

country: Israel

institute: University of Haifa

summary: Environmental contamination from heavy metals poses a global concern for the marine environment, as heavy metals are passed up the food chain and persist in the environment long after the pollution source is contained. Cnidarians, dating back to 700 million years ago, play an important role in shaping marine ecosystems, but environmental pollution profoundly affects their vitality. Among the cnidarians, the sea anemone Nematostella vectensis is an advantageous model for addressing questions in molecular ecology and toxicology as it tolerates extreme environments and its genome has been published. Here we employed a transcriptome-wide RNA-Seq approach to analyze N.vectensis molecular defense mechanisms against four heavy metals: Hg, Cu, Cd and Zn. Altogether, more than 5000 transcripts showed significant changes in gene expression, with Hg having the greatest impact on up-regulating transcripts, followed by Cu, Cd and Zn. We identified, for the first time, co-up-regulation of immediate-early transcription factors such as Egr1, AP1 and NF-κB. Time-course analysis of these genes revealed their early expression as rapidly as one hour after exposure to heavy metals, suggesting that they may complement or substitute for the roles of the metal mediating Mtf1 transcripton factor. We further characterized regulation of a large array of stress-response gene families including Hsp, ABC, CYP members and phytochelatin synthase that may regulate synthesis of the metal-binding phytochelatins instead of the methallothioneins that are absent from Cnidaria genome. This study provides mechanistic insight into heavy-metal toxicity in N.vectensis and sheds light on ancestral stress adaptations.

overall_design: 4 metals were tested in triplicates in comparison to control (4 replicates)

first: Ron

last: Elran

first: Maayan

last: Raam

first: Roey

last: Kraus

first: Vera

last: Brekhman

first: Noa

last: Sher

first: Inbar

last: Plaschkes

first: Vered

last: Chalifa-Caspi


title: Identification of glucocorticoid receptor (GR) binding sites in multiple myeloma

city: Chicago

first: Nancy

last: Krett

state: IL

address: 840 South Wood Street, 740 CSB

department: Medicine

institute: University of Illinois Chicago

summary:
Purpose: The glucocorticoid receptor is widely expressed across mutliple tissues, and yet has very tissue specfic actions.  This is most likely due to the tissue specific chormatin landscape which dictates GR binding.  It identify GR tragets in myeloma as potential therapeutic targets, we have used ChIP-seq to identify myeloma specific GR binding sites.
Methods:  DNA-chromatin complexes were isolated from MM.1S (GR positive) or MM.1RL (GR negative) cells following a 2 hour treatment with either 1 micromolar dexamethasone or vehicle control. Complexes were immunoprecipitated with either a GR specific antibody or an IgG control and the DNA isolated for next generation sequencing.
Results: We identified 8689 high confidence GR binding sites and further correlated those sites to specific genes which are regulated by glucocorticoids through integration with gene expression array data from our lab (submitted to GEO speparately).
Conclusions: Overall, our results indentified nearly 400 genes under the direct transcriptional control of glucocorticoids in myeloma cells.

overall_design: ChIP seq analysis of GR binding in 2 myeloma cell lines after 2 hour treatment with glucocorticoids or vehicle control.

first: Steven

last: Rosen

first: Preethi

last: Gunaratne

first: Cristian

last: Coarfa


title: Expanding the CTD code: methylation of non-consensus Lysine residues marks early transcription in mammalian cells

city: Berlin

first: Elena

last: Torlai Triglia

state: Deutschland

address: Robert Rössle Str 10

department: BIMSB

country: Germany

institute: Max Delbrück Center for Molecular Medicine

summary: Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and co-transcriptional processing of nascent RNA. Extensive phosphorylation of serine residues occurs at the structurally-disordered C-terminal domain (CTD) of the largest RNAPII subunit, which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe for the first time mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation.

overall_design: Genome wide mapping of 2 novel RNAPII post-translational modifications (CTD-K7me1 and CTD-K7me2) in mouse ES cells.

first: Joao

last: Dias

first: Tiago

last: Rito

first: Carmelo

last: Ferrai

first: Alexander

last: Kukalev

first: Hiroshi

last: Kimura

first: Ana

last: Pombo


title: Next Generation Sequencing Facilitates Quantitative Analysis of chromatin loci bound by DDX5 in cultured T helper 17 cell [ChIP-Seq]

status: Public on Dec 21 2015

first: Wendy

last: Huang

address: 540 First Ave Skirball 2-14

institute: NYU School of Medicine

summary:
Purpose: The goals of this study are to identify chromatin loci bound by DDX5 in cultured T helper 17 cell in wildtype and DDX5 deficient background.
Methods: ChIP-seq profiles of 48hrs in vitro cultured T helper 17 cells from wild-type mice were generated by deep sequencing, using Illumina HighSeq. The sequence reads that passed quality filters were mapped by Bowtie2.0.
Results: DDX5 were found to be enriched on a subset of previously characterized RORgt occupied sites on the chromatin.
Conclusions: Our study suggest that a subset of RORgt occupied regions were co-bound by DDX5.

overall_design: DDX5-associated-DNA profiles of 48hr in vitro cultured Th17 from wild type (WT) and DDX5 T cell specific conditional knockout mice were generated by deep sequencing using Illumina HighSeq

first: Dan

last: Littman


title: ChIP-seq analysis of MBD3 in human breast cancer cells

city: RTP

first: Takashi

last: Shimbo

address: 111 TW Alexander Dr.

institute: NIH/NIEHS

summary: Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is a powerful approach to identify the genome-wide localization of protein of interest. However, to perform ChIP-seq experiments for large protein complex, such as Mi-2/NuRD complex, is still challenging. Here, we present high-quality MBD3 ChIP-seq data in human breast cancer cells, MDA-MB-231.

overall_design: MBD3 ChIP-seq experiment was perform using human breast cancer cells, MDA-MB-231. Two bilogical replicates were prepared.

first: Motoki

last: Takaku

first: Paul

middle: A

last: Wade


title: ChIP-seq data from fkh-Gal4::UAS-rib-GFP and sage-Gal4::UAS-rib-GFP mid- through late-stage Drosophila embryos

city: Duluth

first: Joslynn

middle: Shannon

last: Lee

state: MN

address: 1035 University Drive

department: Biomedical Sciences

institute: University of Minnesota Medical School, Duluth campus

summary: Transcription factors, which regulate the spatiotemporal patterns of gene expression during organogenesis, often regulate multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape changes during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the abnormal cell/organ shape in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib that control cell and organ shape, we performed ChIP-seq analysis in embryos driving rib expression specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression through transcriptional activation, repression, or both, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites of bound Rib most likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell shape change in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that a critical component of the SG morphogenetic gene network involving Rib is its autoregulation.

overall_design: chromatin from three independent collections of stage 11 – 16 fkh-Gal4::UAS-rib-GFP embryos and three of sage-Gal4::UAS-rib-GFP embryos were cross-linked at room temperature in 1.8% formaldehyde in 2ml of homogenization buffer (60mM KCl, 15mM NaCl, 15mM HEPES {pH7.6}, 4mM MgCl2, 0.5mM DTT, 0.5% Triton X-100 and cOmplete™ protease inhibitor cocktail {1 tablet per 50ml buffer}). The cross-linked material was resuspended in 0.1%SDS and 0.5% N-lauroylsarcosine in 0.5ml lysis buffer (140mM NaCl, 15mM HEPES {pH 7.6}, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 1% Triton X-100, 0.5mM DTT and cOmplete™ protease inhibitor cocktail {1 tablet per 50 ml buffer}). Chromatin was sonicated thrice at 4°C using the Sonic Dismembrator Model 100 (Fisher Scientific) under the following conditions: power setting 3, 20s ON, 20s OFF. Immediately after sonication, the chromatin extract was stored at -80°C before it was used for immunoprecipitation. Immunoprecipitations were performed using a polyclonal goat anti-GFP antibody (a gift from Kevin White). Immunoprecipitated DNA was prepared for Illumina sequencing using the TruSeq ChIP Sample Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2000 (50 bp reads) according to the manufacturer’s standard protocols. Library preparation and Illumina sequencing were performed at the University of Minnesota Genomics Center.

first: Deborah

last: Andrew

first: Rajprasad

last: Loganathan


title: Next Generation Sequencing Facilitates Quantitative Analysis of RNAs bound by DDX5 and RORgt in cultured T helper 17 cell [ChIRP-Seq]

summary:
Purpose: The goals of this study are to compare RNAs bound by DDX5 and RORgt in cultured T helper 17 cell in wildtype background.
Methods: Th17 mRNA profiles of 48hrs in vitro cultured T helper 17 cells from wild-type mice were generated by deep sequencing, using Illumina HighSeq. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) and TopHat followed by CuffDiff. qRT–PCR validation was performed using SYBR Green assays.
Results: Among the 3444 RefSeq non-coding RNAs and 46449 NONCODE non-coding RNAs, 2533 were found to be expressed in Th17 cells (FPKM>1). 210 of them were enriched in DDX5 pull-down and 119 of them were enriched in RORgt pull-down.
Conclusions: Our study suggest that a subset of 31 ncRNAs were co-enriched in DDX5 and RORgt pull-down.

overall_design: DDX5 or RORgt-associated-RNA profiles of 48hr in vitro cultured Th17 from wild type (WT) mice were generated by deep sequencing using Illumina HighSeq


title: RNA-seq analyses of kdm5 mutant flies and wt flies under normal condition and oxidative stress

first: Julie

last: Secombe

summary: Comparing the transcriptome of wildtype and kdm5 mutant flies in normal conditions revealed a total of 4787 genes that were significantly downregulated and thus require KDM5 for their activation, and 3269 upregulated genes that are normally repressed by KDM5 (p<0.05, FDR <0.05).  Because kdm5 mutants are sensitive to the oxidizer paraquat, we also carried out RNA-seq from wildtype and kdm5 mutant adults in oxidative stress conditions.  Paraquat treatment of wildtype flies lead to the upregulation of 2481, and downregulation of 3103 genes

overall_design: adult mRNA profiles of 1-3-days old wild type (WT) and kdm5 mutant under normal condition and oxitative stress were generated by deep sequencing, using Illumina HisSeq 2000.

first: Xingyin

last: Liu


title: Identifying transcription start sites and active enhancer elements using BruUV-seq

city: Ann Arbor

first: Mats

last: Ljungman

state: Michigan

address: NCRC, B520 Room 1346 2800 Plymouth Rd.

institute: University of Michigan

summary: BruUV-seq utilizes UV light to introduce transcription-blocking DNA lesions randomly in the genome prior to bromouridine-labeling and deep sequencing of nascent RNA. By inhibiting transcription elongation, but not initiation, pre-treatment with UV light leads to a redistribution of transcription reads resulting in the enhancement of nascent RNA signal towards the 5′-end of genes promoting the identification of transcription start sites (TSSs). Furthermore, transcripts associated with arrested RNA polymerases are protected from 3′–5′ degradation and thus, unstable transcripts such as putative enhancer RNA (eRNA) are dramatically increased. Validation of BruUV-seq against GRO-cap that identifies capped run-on transcripts showed that most BruUV-seq peaks overlapped with GRO-cap signal over both TSSs and enhancer elements. Finally, BruUV-seq identified putative enhancer elements induced by tumor necrosis factor (TNF) treatment concomitant with expression of nearby TNF-induced genes. Taken together, BruUV-seq is a powerful new approach for identifying TSSs and active enhancer elements genome-wide in intact cells.

overall_design: Two cell lines were used.  K562 cells were mock-irradiated (control) or UVC-irradiated at two different doses (25 and 100 J/m^2).  HF1 cells were UVC-irradiated (20 J/m^2) in three independent experiments (nfUV4,nfUV3a, and nfUV3b).  In one experiment, HF1 cells were also treated with TNF (10 ng/mL) 1 h prior to UV irradiation (tnfpreUV2, paired with nfUV4).

first: Brian

last: Magnuson

first: Artur

last: Veloso

first: Killeen

last: Kirkconnell

first: Leonardo

last: Andrade-Lima

first: Michelle

last: Paulsen

first: Emily

first: Karan

last: Bedi

first: Jayendra

last: Prasad

first: Thomas

last: Wilson


title: Expression profiling for mouse embryonic stem cells deficient for Smad1 and Smad5 or for Bmp activated subpopulations.

first: Ruben

last: Dries

address: 360 Longwood Ave

department: Computational Biology

institute: DFCI

summary: In this study we determine the transcriptional profile by RNAseq of mESC in the absence of Smad1 and Smad5 and in subpopulation of mESC with different levels of BMP-SMAD activation.

overall_design:
Transcriptome analysis using RNAseq was performed on 3 biological replicates of BRE negative and positive mESC subpopulations, which were collected in pairs at 3 different times.
Transcriptome analysis using RNAseq was performed on Smad1/5 floxed (FL) and knockout (KO) mESC. Two different parental cell lines were used.
For each parental cell line we analyzed one Smad1/5 FL sample and two Smad1/5 KO samples, resulting in respectively two and four biological replicates for the FL and KO conditions.

first: Maria

last: Gomes Fernandes

first: Susana

last: Chuva de Sousa Lopes


title: Neurospora MUS-30 is an LSH/DDM1 homolog required for normal genome maintenance

city: Athens

first: Robert

last: Schmitz

state: GA

address: B416 Davison Life Sciences

institute: University of Georgia

summary: LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to the DNA damaging agent MMS (methyl methanesulfonate). MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued MMS-hypersensitivity of Dmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Dmus-30 is also partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Dmus-30 strains. It was reported that mammalian LSH is required for efficient double strand break (DSB) repair. We found that MUS-30-deficient cells were not defective for DSB repair, and we observed a negative genetic interaction between Dmus-30 and Dmei-3, the Neurospora RAD51 homolog required for homologous recombination. These data are consistent with a role for MUS-30 that is independent of DSB repair. Our findings demonstrate that LSH/DDM1 enzymes are key regulators of genome stability in eukaryotes.

overall_design: crf5-1 isolates (two replicates each from the F1 and F2 generation) were grown in Vogel's minimal medium for 48 hours. As a control, two replicates of the wildtype strain were grown under identical conditions.


title: Genome-wide Ribbon occupancy and gene expression profiling of wildtype and ribbon mutant Drosophila mid through late stage embryos

city: Baltimore

first: Deborah

last: Andrew

state: MD

address: 725 N. Wolfe St., Hunterian G-1

department: Cell Biology

institute: Johns Hopkins University School of Medicine

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

overall_design: Refer to individual Series


title: Single-cell RNA sequencing of aspirates from cortical neurons after patch clamp recording

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

summary: We obtained full transcriptome data from single cortical neurons after whole-cell patch-clamp recording (termed “Patch-seq”). By applying “Patch-seq” to cortical neurons, we reveal a close link between biophysical membrane properties and genes coding for neurotransmitter receptors and channels, including well-established and hitherto undescribed subtypes.

overall_design: RNA sequencing was performed on a total of 83 individual cells

first: Fuzik

last: Janos

first: Zeisel

last: Amit

first: Linnarsson

last: Sten

first: Harkany

last: Tibor


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type, DDX5 knockout, RORc knockout, RmrpG262T knockin T helper 17 cell Transcriptomes [RNA-seq]

first: Wendy

last: Huang

summary:
Purpose: The goals of this study are to compare in vitro polarized T helper 17 cell transcriptome profiling (RNA-seq) in different genetic background.

Methods: Th17 mRNA profiles of 96hrs in vitro cultured T helper 17 cells from wild-type and mutant mice were generated by deep sequencing, using Illumina RapidRun. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) and TopHat followed by CuffDiff. qRT–PCR validation was performed using SYBR Green assays.

Results: Among the 325 genes that were significantly dysregulated in DDX5-deficient T cells, approximately 40% were previously identified as RORγt targets in Th17 cells. 96 RORγt-dependent Th17 cell genes co-regulated by Rmrp together with DDX5.

Conclusions: Our study suggest that the DDX5-Rmrp axis is critical for expression of a critical subset of the RORγt transcriptional targets in Th17 cells.

overall_design: mRNA profiles of 96hr in vitro cultured Th17 from wild type (WT) and mutant mice were generated by deep sequencing using Illumina RapidRun.

first: Dan

last: Littman


title: KDM5 occupancy and expression profiling of kdm5 mutant flies and wt flies under normal condition and oxidative stress

first: Julie

last: Secombe

address: 1300 Morris park ave. Ullmann 809

institute: Albert einstein college of medicne


title: DNA methylation analysis for mouse embryonic stem cells deficient for Smad1 and Smad5 or for Bmp activated subpopulations.

summary: In this study we determine the profile of DNA methylation by RRBseq of mESC in the absence of Smad1 and Smad5 and in subpopulation of mESC with different levels of BMP-SMAD activation. We observed a general loss of DNA methylation associated with low or absent BMP-SMAD signalling in mESCs.

overall_design:
DNA methylation analysis using RRBS was performed on 3 biological replicates of BRE negative and positive mESC subpopulations, which were collected in pairs at 3 different times.
DNA methylation analysis using RRBS was performed on Smad1/5 floxed (FL) and knockout (KO) mESC. Two different parental cell lines were used.
For each parental cell line we analyzed one Smad1/5 FL sample and two Smad1/5 KO samples, resulting in respectively two and four biological replicates for the FL and KO conditions.


title: Telomere Binding Protein TRB1 Preferentially Associates with Promoters of Translation Machinery genes

city: Brno

first: Jan

last: Hapala

address: Kamenice 5

country: Czech Republic

institute: Masaryk University - CEITEC

summary:
Background: Recently we characterised TRB1, a protein from a single-myb-histone family, as a structural and functional component of telomeres in Arabidopsis thaliana. TRB proteins, besides their ability to bind specifically telomeric DNA using their N-terminally positioned myb-like domain of the same type as in human TRF1 or TRF2 shelterin proteins, possess also histone-like domain which is involved in protein-protein interactions e.g., with POT1b. We set to investigate the genome-wide localization pattern of TRB1 to reveal its preferential mode of binding to chromatin in vivo and its potential functional roles in the genome-wide context.
Results: Our results demonstrate that in addition to its roles at telomeres, TRB1 is preferentially associated with promoter regions of genes involved in ribosome biogenesis. This preference coincides with a frequent occurrence of telobox motifs in the upstream regions of genes in this category, but is not restricted to the telobox presence.
Conclusions: We conclude that TRB1, in addition to its preferential telomeric localization, shows a specific genome-wide distribution pattern suggesting its role in regulation of genes involved in biogenesis of translational machinery.

overall_design: TRB1 DNA-binding activity investigated in 2 biological replicates (2 immunoprecipitation approaches) with controls; with technical replicates.

first: Petra

last: Procházková Schrumpfová

first: Ivona

last: Vychodilová

first: Šárka

last: Schořová

first: Vojtěch

last: Dvořáček

first: Jiří

last: Fajkus


title: Gene expression data of human peripheral blood-derived cultured mast cells

city: Tsukuba

first: Akira

last: Shibuya

state: Ibaraki

address: 1-1-1 Tennodai

country: Japan

institute: University of Tsukuba

summary: Mast cell (MC) activation contributes considerably to immune responses, such as host protection and allergy. Cell surface immunoreceptors expressed on MCs play an important role in MC activation. Although various immunoreceptors on MCs have been identified, the regulatory mechanism of MC activation is not fully understood. To understand the regulatory mechanisms of MC activation, we used gene expression analyses of human MCs to identify a novel immunoreceptor expressed on MCs. We found that Tek, which encodes Tie2, was preferentially expressed in the MCs of humans.

overall_design: Total RNA of peripheral blood (PB)-derived cultured MCs and other PR-derived cells were extracted by using an RNeasy Mini kit (Qiagen, Hilden, Germany), and a Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA) was used to remove ribosomal RNA. RNA-seq was performed according to the protocol described in the SOLiD Total RNA-Seq Kit (Life Technologies, Carlsbad, CA). The library was subjected to emulsion PCR (SOLiD™ EZ Bead™ Emulsifier kit, Life Technologies) to generate clonal DNA fragments on beads, followed by bead enrichment (SOLiD™ EZ Bead™ Enrichment kit, Life Technologies). Enriched template beads were sequenced on a SOLiD 5500xl sequencer as single-end, 75-bp reads (Life Technologies). The SOLiD 5500xl output reads were aligned against the human genome reference sequence (hg19) by using LifeScope version 2.5.1 (Life Technologies) to generate BAM files, and subsequent data analysis was performed in Avadis NGS (Strand Scientific Intelligence Inc., San Francisco, CA).

first: Kazumasa

last: Kanemaru

first: Emiko

last: Noguchi

first: Takahiro

last: Tokunaga

first: Kei

last: Nagai

first: Satoko

last: Tahara-Hanaoka


title: Genome wide DNA methylation sequencing of ischemic skeletal muscle from normal, hyperlipidemic and type 2 diabetic mice

city: Kuopio

first: Mohan

last: Babu

address: Neulaniementie 2

department: Biotechnology and Molecular Medicine

country: Finland

institute: A.I.Virtanen Institute, University of Eastern Finland

summary:
Objective: To define the role of epigenetics, particularly DNA methylation in adaptive vascular growth in hyperlipidemic and type 2 diabetic mouse models of hind limb ischemia
Methods: Unilateral hindlimb ischemia was induced by ligating femoral artery proximal to the bifurcation of superficial and deep femoral artery. DNA was isolated from ischemic muscles collected at day 7 after ischemia induction using DNeasy Tissue kit (Qiagen). 5 µg of genomic DNA was sheared into small fragments with a mean size of 150 bp by using a Covaris™ S2 sonicator System. Quality of the fragmentation was analyzed with Bioanalyzer. Fragmented DNA samples were used for preparation of DNA fragment libraries and sequenced on the SOLiD4 sequencing instrument on one flow cell for 50 bp reads. The sequencing reads were mapped to mus musculus genome build mm9. Data was analyzed by using Bioscope. The samples were normalized with the MEDIPS package in R/B Bioconductor. The analysis of CpG methylation was done primarily in the proximal promoter regions encompassing a region of –1kb upstream of the transcription start site (TSS) and +500 bp downstream of the TSS. Comparisons were performed between hyperlipidemic versus controls and diabetic versus controls to detect differentially methylated regions. R package Limma was used for performing the statistical testing between the groups.
Results: When visualizing the whole normalized data the samples did not cluster according to the sample groups. Especially two samples from hyperlipidemic and diabetic ischemic muscles differed clearly from the rest of the samples. To detect the differentially methylated genes, stringent thresholds for p-values and fold change values were chosen to list a reasonable number of genes. Upon filtering, significant differences in the methylation patterns of the sample groups were observed. More importantly, when clustering only the filtered genes, the samples clustered clearly according to the sample groups giving evidence of condition-dependent behavior. Using a threshold of methylation fold change of >1.2 and p value <0.05, we identified 397 and 446 genes to be hypomethylated in hyperlipidemic and diabetic ischemic muscles respectively compared to controls. There were 46 genes commonly shared, but still having a unique pattern of hypomethylation in 371 and 394 genes in hyperlipidemic and diabetic ischemic muscles respectively compared to controls. Similarly, there were 371 and 394 genes hypermethylated in hyperlipidemic and diabetic ischemic respectively compared to controls. Interestingly, we found 264 genes to be commonly hypermethylated, whereas 107 and 130 genes were uniquely hypermethylated in hyperlipidemic and diabetic ischemic muscles respectively. Thus, proximal promoter methylation suggested a shared, yet distinct pattern of DNA methylation in ischemic muscles of hyperlipidemic and type 2 diabetic mice compared to controls. Out of 397 genes that were hypomethylated in hyperlipidemic ischemic muscle, 68 genes were shown to be upregulated in ‘proinflammatory M1 macrophages’ as shown by recent studies. Similarly, out of the 371 hypermethylated genes 93 genes were shown to be upregulated in ‘anti-inflammatory and proangiogenic M2 macrophages’ as described recently. Out of 446 hypomethylated genes in diabetic ischemic muscle, 65 genes were shown to be upregulated in ‘proinflammatory M1 macrophages’ as shown recently. Similarly, out of 394 hypermethylated genes 105 genes were specifically upregulated in ‘anti-inflammatory and proangiogenic M2 macrophages’ as shown recently. qRT-PCR analysis suggested an inverse relationship between proximal promoter hypermethylation and mRNA expression in a subset of M2 macrophage specific genes in hyperlipidemic and type 2 diabetic ischemic muscles compared to control ischemic muscles.
Conclusions: Our results suggest a role of epigenetics particularly proximal promoter DNA methylation in macrophage polarization and their contribution to angiogenesis and tissue repair in hyperlipidemic and type 2 diabetic mouse models of hind limb ischemia. Epigenetics at the level of DNA methylation may act as a deciding factor in promoting a pro or anti-inflammatory phenotype of macrophages critical in cardiovascular diseases.

overall_design: Ischemic skeletal muscle DNA methylation sequencing of triplicate samples from C57BL/6J (WT) mice, hyperlipidemic mice (LDLR-/-ApoB100/100 , C57BL/6J background) and type 2 diabetic mice (IGF-II/LDLR-/-ApoB100/100 , C57BL/6J background) using SoliD4 sequencing platform

first: Seppo

last: Yla-Herttuala


title: Differential promoter methylation of macrophage genes correlates with impaired vascular growth in ischemic muscles of hyperlipidemic and type 2 diabetic mice


title: Genome wide promoter methylation analysis of mouse macrophages and endothelial cells isolated from ischemic hindlimbs using RRBS sequencing

summary:
Purpose: The goal of this study was to compare the genome-wide promoter methylation alterations in macrophages and endothelial cells during hindlimb ischemia among normal, hyperlipidemic and type-2 diabetic mice.
Methods: Unilateral hindlimb ischemia was induced by ligating femoral artery proximal to the bifurcation of superficial and deep femoral artery in mice deficient of LDL receptor and expressing only apolipoprotein B100 (LDLR-/-ApoB100/100, C57BL/6J background) (The Jackson  Laboratory, Bar Harbor,USA) and mice with β-cell specific over-expression of insulin-like growth factor-2 in atherosclerotic background (IGF-II/LDLR-/-ApoB100/100, C57BL/6J background) with type 2 diabetic features on high-fat diet (TD 88173, Harlan Teklad: 42% of calories from fat and 0.15% from cholesterol, no sodium cholate) 8 weeks prior to surgery and continued throughout the study 1. C57BL/6J (WT) mice fed with regular chow-diet (R36, Lactamin) served as controls. All animals were aged between 20 to 24 weeks at the time of hindlimb operations. For sorting macrophages from ischemic muscles, ischemic gastrocnemius muscles were minced and enzymatically dissociated using a cocktail containing 450 U/mL Collagenase I, 125 U/mL Collagenase XI, 60 U/mL DNAseI, and 60 U/mL hyaluronidase (Sigma Aldrich) for 1 h at 37°C. The cells were then counted and divided into CD31+ve and CD31-vefractions using CD31 magnetic bead enrichment (Miltenyi Biotec). For macrophage sorting CD31-ve fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with FITC conjugated rat anti-mouse F4/80 antibody (Serotec) for 20 minutes at 4˚C. For endothelial sorting CD31+ fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with APC conjugated rat anti-mouse CD31 antibody (BD-pharmingen) and FITC conjugated rat anti-mouse CD45 ((BD-pharmingen) for 20 minutes at 4˚C. FACS sorting was performed on FACS AriaIII (BD Biosciences). Genomic DNA was isolated from FACS sorted macrophages and endothelial cells using AllPrep DNA/RNA/Protein Mini Kit (Qiagen Finland, Helsinki, Finland) according to manufacturer's instructions.
Results: The sample similarity as assessed by Pearson’s correlation matrix and Hierarchial clustering showed high correalation among macrophages, as well as endothelial cells. There was a clear clustering of macrophages and endothelial cells as evidence by their CpG methylation clustering, furthermore macrophages from HL and T2DM mice showed clear clustering compared to control macrophages. Differential methylation analysis of RRBS methylation data from macrophages and endothelial cells was performed using Methylkit. Using a threshold of adjusted p value (Q) <0.05 and percentage methylation difference of >5%, we identified 198 and 272 genes whose promoters were hypomethylated in HL and T2DM macrophages. Similarly, there were 102 and 136 gene promoters were hypermethylated in HL and T2DM macrophages, respectively compare to control macrophages. Thus, proximal promoter methylation suggested that HL and T2DM have convergent influences on the proximal promoter methylation of numerous macrophage specific genes. In order to find out whether these genes with differential methylated promoters were differentially expressed at  mRNA expression level in purified macrophages, we further compared our data with the GEO datasets as above. Of the 198 genes with promoter hypomethylation in HL macrophages 72 genes were suggested to be upregulated in M1- Mϕs; whereas, of the 102 genes with promoter hypermethylation, 51 genes were suggested to be upregulated in M2- Mϕs. Similarly, out of 272 genes with differentially methylated promoters in T2DM macrophages 88 genes were suggested to be upregulated in M1-Mϕs; whereas, out of 136 genes with promoter hypermethylation 60 genes were suggested to be upregulated in M2- Mϕs. Thus a significant promoter hypomethylation of M1-Mϕ and hypermethylation of M2-Mϕ genes suggested the predominance of proinflammatory M1-Mϕs in ischemic muscles of HL and T2DM compared to M2-Mϕs in control mice.
Conclusions: We found significant promoter hypomethylation of genes typical for proinflammatory M1-Mϕs and hypermethylation of anti-inflammatory, proangiogenic M2-Mϕ associated genes in HL and T2DM ischemic muscles. Epigenetic alterations  skewing Mϕ phenotype towards proinflammatory as opposed to anti-inflammatory, proangiogenic and tissue repair phenotype may contribute to impaired adaptive vascular growth in these pathological conditions.

overall_design: Macrophages and endothelial whole genome DNA methylation was performed in triplicates (Each sample was pooled from 3-4 mice) by RRBS Sequencing approach using Illumina HiSeq 2500. qRT–PCR validation was performed using TaqMan assays.

last: Ylä-Herttuala


title: Engineering an allosteric transcription factor to respond to new ligands

status: Public on Dec 20 2015

first: Alexander

last: Garruss

state: MA

address: 77 Avenue Louis Pasteur, Rm 238

institute: Harvard Medical School

summary: Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. A major class of regulatory proteins is microbial allosteric transcription factors (aTFs), but aTF–inducer pairs are currently limited by those that naturally occur. Altering inducer specificity in these proteins is difficult because mutations that affect inducer binding may also disrupt allostery. Here, we engineer an aTF, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol or sucralose. We employ computational protein design, single-residue saturation mutagenesis, or random mutagenesis, along with multiplex assembly, and identify initial hits via a two-stage enrichment screen. Following activity maturation, we identify LacI variants with specificity to and induction by these new inducers comparable to that of wild-type LacI and its inducer, IPTG. The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.

overall_design: Identification of E. coli LacI protein variants depleted after negative selection by next-generation sequencing.

first: Noah

last: Taylor

first: Srivatsan

last: Raman

first: George

last: Church


title: Mouse embryonic stem cells deficient for Smad1 and Smad5 or for Bmp activated subpopulations.

first: Ruben

last: Dries


title: Zscan10 is dispensable for maintenance of pluripotency in mouse embryonic stem cells

city: Kobe

first: Hitoshi

last: Niwa

state: Hyogo

address: 2-2-3 Minatojima-minamimachi,Chuo-ku

department: Center for Developmental Biology

institute: RIKEN

summary: Zinc finger and SCAN domain-containing 10 (Zscan10, also known as Zfp206) encodes a transcription factor that has been reported to be involved in the maintenance of pluripotency in mouse embryonic stem (ES) cells. Here we generated inducible knockout ES cells for Zscan10 using the Cre-loxP system and analyzed its function. We succeeded in establishing Zscan10-null ES cells and confirmed their pluripotency by the generation of chimeric embryos. Our results clearly indicate that Zscan10 is dispensable for the ability of self-renewal and differentiation in ES cells.

overall_design: An inducible knockout ES cell line of Zscan10 with the Cre-loxP system was generated and gene expression was measured without Zscan10 deletion or constitutive knocked out cells. Each sample was prepared in triplicate.

first: Mariko

last: Yamane

first: Setsuko

last: Fujii

first: Satoshi

last: Ohtsuka


title: microRNA sequencing in rat cardiac fibroblasts and fibroblast-derived exosomes

status: Public on Dec 19 2015

city: Hannover

first: Claudia

last: Bang

address: Carl-Neuberg-Straße 1

institute: Medical School Hannover

summary: We found that cardiac fibroblasts produce and secrete exosomes. miRNA profiling and TaqMan qRT-PCR experiments  identified miR-21 expression to be higher in cardiac fibroblasts compared to those of miR-21*, whereas in exosomes miR-21* expression was higher compared to miR-21. The purpose of the study was to validate these findings by miRNA sequencing in cardiac fibroblasts and fibroblasts-derived exosomes.

overall_design: Neonatal rat cardiac fibroblasts were cultured in DMEM + 1% exosome-depleted FBS for 48h. Conditioned medium was collected and exosomes were purified by several centrifugation and filtration steps, following ultracentrifugation. Afterwards total RNA from cardiac fibroblasts and exosomes was isolated for miRNA sequencing.

first: Rashi

last: Halder


title: Cause and consequence of tethering a sub-TAD to different nuclear compartments

city: Utrecht

first: Geert

last: Geeven

state: Utrecht

address: Uppsalalaan 8

department: Biomedical Genomics

country: Netherlands

institute: Hubrecht Institute

summary: Detailed genomic contact maps have revealed that chromosomes are composed of  developmentally stable topologically associated domains (TADs) and more flexible  sub-TADs. These domains reside in active and inactive nuclear compartments, but  cause and consequence of compartmentalization are largely unknown. Here, we  combined lacO/lacR binding platforms with allele-specific 4C technologies to track their  precise position in the three-dimensional genome upon recruitment of NANOG,  SUV39H1 or EZH2. We observed locked genomic loci resistant to spatial repositioning  and unlocked loci that could be repositioned to different nuclear sub-compartments  with distinct chromatin signatures. Focal protein recruitment caused the entire sub-  TAD, but not surrounding regions, to engage in new genomic contacts. Compartment  switching was uncoupled from gene expression changes and enzymatically modifying  histones per se was insufficient for repositioning. Collectively this suggests that transassociated  factors determine three-dimensional compartmentalization independent of  their cis-effect on local chromatin composition and activity.

overall_design: 4C-seq was performed on a range of viewpoints in 129/Sv;C57BL/6 embryonic stem cells carying a lacO array in chromosome 8 and 11.

first: Patrick

last: Wijchers

first: Peter

last: Krijger

first: Wouter

last: de Laat


title: Genome-wide analysis of 6 month old hippocampal gene expression in Ogg1- and/or Mutyh-deficent mice

city: Trondheim

first: Katja

last: Scheffler

address: Edvard Griegs Gate 8

department: Department of Neuromedicine and Movement Sciences

country: Norway

institute: Norwegian University of Science and Technology

summary: Oxidative DNA damage has been associated with cognitive decline. The Ogg1 and Mutyh DNA glycosylases cooperate to prevent mutations caused by 8-oxoG, a major premutagenic oxidative DNA base lesion. Here, we have examined behavior and cognitive function in mice deficient of these glycosylases. We found that Ogg1-/-Mutyh-/- mice were more active and less anxious and that their learning ability was impaired. In contrast, Mutyh-/- mice showed moderately improved memory compared to WT. There was no change in genomic 8-oxoG levels, suggesting that Ogg1 and Mutyh play minor roles in global repair in adult brain. Notably, transcriptome analysis of hippocampus revealed that differentially expressed genes in the mutant mice belong to pathways known to be involved in anxiety and cognitive function. Thus, beyond their involvement in DNA repair, Ogg1 and Mutyh modulate cognitive function and behavior, and related hippocampal gene expression, suggesting a novel role for 8-oxoG in regulating adaptive behavior.

overall_design: The mRNA profiles from hippocampus of WT, Ogg1-/-, Mutyh-/- and Ogg1-/- Mutyh-/- C57BL/6 mice at 6month of age were generated by RNA sequencing using Illumina Hiseq 2000

first: Monica

last: Bjørge

first: Gunn

last: Hildrestrand

first: Rajikala

last: Suganthan

first: Veslemøy

last: Rolseth

first: Anna

last: Kusnierczyk

last: Rowe

first: Cathrine

middle: B

last: Vågbø

first: Susanne

last: Vetlesen

first: Lars

last: Eide

first: Geir

last: Slupphaug

first: Yusaku

last: Nakabeppu

first: Timothy

middle: W

last: Bredy

first: Arne

last: Klungland

first: Magnar

last: Bjørås


title: Gene expression of M. acetivorans producing ANME-1 Mcr during growth on methane vs. growth on methanol

city: State College

first: Thomas

middle: K.

last: Wood

state: PA

address: Room 161 Fenske Laboratory, University Park

department: Chemical Engineering & Biochemistry and Molecular Biology

institute: Pennsylvania State University

summary: Our goal is to convert methane efficiently into liquid fuels that may be more readily transported. Since aerobic oxidation of methane is less efficient, we focused on anaerobic processes to capture methane, which are accomplished by anaerobic methanotrophic archaea (ANME) in consortia. However, no pure culture capable of oxidizing and growing on methane anaerobically has been isolated. In this study, Methanosarcina acetivorans, an archaeal methanogen, was metabolically engineered to take up methane, rather than to generate it. To capture methane, we cloned the DNA coding for the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable archaeal organism from a Black Sea mat into M. acetivorans to effectively run methanogenesis in reverse. The engineered strain produces primarily acetate, and our results demonstrate that pure cultures can grow anaerobically on methane.

overall_design: Differential gene analysis of two growth conditions (three biological replicates each) was performed: (i) M. acetivorans/pES1-MATmcr3 grown on methane and (ii) M. acetivorans/pES1-MATmcr3 grown on methanol. All starter cultures (200 mL) were grown on methanol for 5 days, and harvested by centrifugation. Cell pellets were washed three times with HS medium, and resuspended using 5 mL HS medium, 2 µg/mL puromycin, and 0.1 mM FeCl3. For condition (i), methane was filled into the headspace of the cultures. For condition (ii), 150 mM methanol was added. All cultures were incubated at 37C for 5 days, followed by rapid centrifugation in the presence of 50 µL RNAlater solution (Ambion, Austin, TX) per mL of culture. Total RNA was isolated using RNeasy Mini kit (Qiagen, Valencia, CA) were then digested with terminator 5’-phosphate-dependent exonuclease (Epicentre, Madison, WI) to partially remove ribosomal RNA. Digested RNA were cleaned up using AgenCourt RNAClean XP beads (AgenCourt Bioscience, Beverly, MA) and used for cDNA library construction using the TruSeq Stranded mRNA Library kit (Illumina). Pooled and barcoded cDNA library was then sequenced on a HiSeq sequencing platform (Illumina). Obtained reads were mapped to the reference genome of M. acetivorans (Genbank accession NC_003552.1) using STAR. The mapped reads were assembled using Cufflink v2.2.1 to identify potential novel transcripts. Assembled, unannotated novel transcripts for all the strains were combined with the list of known genes. Differential expression of genes and potential novel transcripts were determined using Cuffdiff at a significance cutoff at q < 0.07 with a false discovery rate of 0.05. Expression levels of gene transcripts are expressed as fragments per kilobase of transcript per million mapped fragments (FPKM), and expression changes are determined by the ratio of FPKM of culture replicates grown on methane to FPKM of culture replicates grown on methanol.

first: Valerie

last: Soo

first: Michael

last: McAnulty

first: Arti

last: Tripathi

first: Fayin

last: Zhu

first: Limin

last: Zhang

first: Emmanuel

last: Hatzakis

first: Philip

last: Smith

first: Saumya

last: Agrawal

first: Hadi

last: Nazem-Bokaee

first: Saratram

last: Gopalakrishnan

first: Howard

last: Salis

first: James

middle: G

last: Ferry

first: Costas

last: Maranas

first: Andrew

last: Patterson


title: Human RNase L Tunes Gene Expression by Selectively Destabilizing the MicroRNA-Regulated Transcriptome

city: Princeton

first: Sneha

last: Rath

state: NJ

address: Lewis Thomas Laboratory

department: Molecular Biology

institute: Princeton University

summary:
Purpose: The goal of this study was to map the pathway of mRNA decay by human RNase L

Methods: Total RNA was extracted (RNeasy kit, Qiagen). RNA integrity was verified by an RNA 6000 Nano Chip, using BioAnalyzer and 2100 Expert software (Agilent Technologies). The mRNA was enriched by oligo-dT pulldown from total RNA, followed by fragmentation, adapter ligation, PCR amplification, and finally sequencing on Illumina HiSeq 2000 platform. For sequencing introns, the oligo-dT pulldown step was replaced with Ribo-Zero rRNA removal (Illumina). Sequencing reads were mapped to the human genome hg19 using TopHat 2  set to map stranded reads with default parameters. Mapped read counts were obtained using HTseq-count run in union mode.

Results: We developed an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and non-targets. We show that this RNase L-dependent decay (RLDD) selectively affects transcripts regulated by miR-17/miR-29/miR-200 and other microRNAs that function as suppressors of mammalian cell adhesion and proliferation. RNase L mimics the effects of these microRNAs and acts as a suppressor of proliferation and adhesion in mammalian cells.

Conclusions: Our data suggest that RLDD serves to establish an anti-proliferative state via destabilization of the microRNA-regulated transcriptome.

overall_design: Human mRNA profiles from HeLa, T47D and HAP1 cells were generated by deep sequencing using Illumina Illumina HiSeq 2000.

first: Jesse

last: Donovan

first: Gena

last: Whitney

first: Alisha

last: Chitrakar

first: Wei

last: Wang

first: Alexei

last: Korennykh


title: Effect of Ets1 knockdown in the P5424 thymic cell line

city: Freiburg

first: Pierre

middle: Daniel

last: Cauchy

state: Baden-Württemberg

address: Breisacher Str. 115

department: Zentrum für Translationale Zellforschung

institute: University Medical Center Freiburg

summary: We performed ChIP-Seq for Ets1 and histone modifications in P5424 thymic cells, without and with Ets1 knockdown via shRNA. Overall, we find that loss of Ets1 results in specific, higher occupancy of H3K4me1-marked nucleosomes at the Ets1 binding site, but not H3K4me3 nucleosomes. We verified the specificity of this mechanism as Ets1-dependent by also computing H3K4me1 and 3 nucleosome occupancy in hypersensitive, Ets1-depleted sites. This effect was also found in primary WT DN+DP Ets1 sites. Overall, this suggests that Ets1 induces chromatin remodeling and that its loss increases H3K4me1 nucleosome occupancy in bound sites, with levels of H3K4me3 comparatively unchanged, thus reducing the activation status of these enhancers

overall_design: Genome-wide analysis via ChIP-Seq for Ets1, H3K4me1 and H3K4me3 in WT, sh-scramble and sh-Ets1 the P5424 thymic cell line

first: Ahmad

last: Maqbool

first: Laurent

last: Vanhille

first: Joaquin

last: Zacarias-Cabeza

first: Frederic

last: Koch

first: Romain

last: Fenouil

first: Angelica

last: Santana

first: Aurélien

last: Griffon

first: Jean

last: Imbert

last: Ferrier

first: Salvatore

last: Spicuglia

first: Jean-Christophe

last: Andrau


title: Next Generation Sequencing Facilitates Quantitative Analysis of RNAs bound by DDX5 and RORgt in cultured T helper 17 cell [RIP-seq]

city: New York

first: Wendy

last: Huang

address: 540 First Ave Skirball 2-14

institute: NYU School of Medicine

summary:
Purpose: The goals of this study are to compare RNAs bound by DDX5 and RORgt in cultured T helper 17 cell in wildtype background.

Methods: Th17 mRNA profiles of 48hrs in vitro cultured T helper 17 cells from wild-type mice were generated by deep sequencing, using Illumina HighSeq. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) and TopHat followed by CuffDiff. qRT–PCR validation was performed using SYBR Green assays.

Results: Among the 3444 RefSeq non-coding RNAs and 46449 NONCODE non-coding RNAs, 2533 were found to be expressed in Th17 cells (FPKM>1). 210 of them were enriched in DDX5 pull-down and 119 of them were enriched in RORgt pull-down.

Conclusions: Our study suggest that a subset of 31 ncRNAs were co-enriched in DDX5 and RORgt pull-down.

overall_design: DDX5 or RORgt-associated-RNA profiles of 48hr in vitro cultured Th17 from wild type (WT) mice were generated by deep sequencing using Illumina HighSeq

first: Dan

last: Littman


title: Genome-wide mapping of retinoblastoma (Rb) binding sites in prostate cancer cell lines VCaP and LNCaP

city: Boston

first: Dong

last: Han

address: 100 Morrissey Blvd

department: Biology-CPCT

institute: Umass Boston

summary: Although well characterized as a transcriptional activator, androgen receptor (AR) can also function as a direct transcriptional repressor in prostate cancer cells.  The major targets of the AR repressive function are genes mediating DNA synthesis. In this study, we found that AR was recruited to the majority of these DNA synthesis genes and rapidly repressed their transcription. This direct AR mediated repression was enhanced in prostate cancer cells expressing higher levels of AR, and was mediated by recruitment of hypophosphorylated retinoblastoma protein (Rb).

overall_design: Examination of Rb binding in 4 hours DHT treated prostate cancer cell lines VCaP and LNCaP

first: Shuai

last: Gao

first: Changmeng

last: Cai


title: Beauveria bassiana SNF1 (BbSNF1) regulating genes involved in devolopment of blastospore

city: Hangzhou

first: Shenghua

last: Ying

state: Zhejiang

address: Yu-hang-tang Road 688

institute: Zhejiang University

summary: Gene BbSNF1 played crucial roles in mediating devolopment of blastospore, a determinant to the virulence of fungal entomopathogens. We characterized for the first time that disruption of BbSNF1 reduced  blastospore yield in culture broth using peptone as single nitrogen source. The global transcriptome  involved in the response to the thermal stress was analyzed by using high throughput sequencing (RNA-Seq). Our transcriptional profiles revealed that numerous differentially expressed genes (DEGs), of which involved in metabolism, cell transport and cell rescue, were significantly involved in deveopment of fungal blastospore.

overall_design: Total RNA obtained from BbSNF1 disruption mutant were compared to that of WT strain in the indicated culture medium.

first: Sheng-Hua

first: Ming-Guang

last: Feng


title: Loss of motoneuron-specific microRNA-218 causes systemic neuromuscular failure [miRNA-seq]

status: Public on Dec 18 2015

city: La Jolla

first: Neal

last: Amin

state: CA

address: 10010 N Torrey Pines Road

department: Gene Expression Laboratory

institute: Salk Institute for Biological Studies

summary: We investigated microRNA expression in motoneurons by performing small RNA sequencing of fluorescence-activated cell sorting (FACS)-isolated motoneurons labelled with the Hb9:gfp transgenic reporter and Hb9:gfp negative non-motoneurons including spinal interneurons.  We find that one microRNA, microRNA-218, is highly enriched and abundantly expressed in motoneurons.  Furthermore, we find that miR-218 is transcribed from alternative, motoneuron-specific alternative promoters embedded within the Slit2 and Slit3 genes by performing RNA sequencing of FACS-isolated motoneurons and a dissected embryonic floor plate cells which served as a control.  Next, we performed RNA sequencing of FACS-isolated wild type (WT) motoneurons and motoneurons lacking miR-218 expression (218DKO motoneurons), and find that a large set of genes (named 'TARGET218' genes) with predicted miR-218 binding sites are de-repressed in the absence of miR-218 expression.  Finally, we examine the expression of TARGET218 genes in other neuronal subpopulations by FACS-isolating  V1, V2a, and V3 interneurons expressing Cre-inducible fluorescent reporters and performing RNA sequencing.  We find that the TARGET218 network of genes is depleted in wild-type motoneurons versus these interneuron types.  Additionally, these genes are expressed at similar levels in 218DKO motoneurons compared with interneuron subtypes, suggesting that this genetic network.

overall_design: Examination of miRNA expression in spinal neuron subpopulations.

first: Samuel

last: Pfaff


title: Loss of motoneuron-specific microRNA-218 causes systemic neuromuscular failure

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

overall_design: Refer to individual Series


title: Testing the Kinship Theory of Intragenomic Conflict in Honey Bees (Apis mellifera)

city: University Park

first: David

middle: Asher

last: Galbraith

state: Pennsylvania

address: 19a Chemical Ecology Lab

institute: Penn State University

summary: Sexual reproduction brings genes from two parents – matrigenes and patrigenes – into one individual. These genes, despite being unrelated, should show nearly perfect cooperation because each gains equally through production of offspring.  However, an individual’s matrigenes and patrigenes can have different probabilities of being present in other relatives, so that kin selection could act on them differently.  Such intragenomic conflict could be implemented by partial or complete silencing (imprinting) of an allele by one of the parents. Evidence supporting this theory is seen in offspring-mother interactions, with patrigenes favoring acquisition of more of the mother's resources if some of the costs fall on half siblings who do not share the patrigene.  The kinship theory of intragenomic conflict is little tested in other contexts, but it predicts that matrigene-patrigene conflict may be rife in social insects.  We tested the hypothesis that honey bee worker reproduction is promoted more by patrigenes than matrigenes by comparing across 9 reciprocal crosses of two distinct genetic stocks. As predicted, hybrid workers show reproductive trait characteristics of their paternal stock, indicating enhanced activity of the patrigenes on these traits, greater patrigenic than matrigenic expression, and significantly increased patrigenic biased expression in reproductive workers.  These results support both the general prediction that matrigene-patrigene conflict occurs in social insects and the specific prediction that honey bee worker reproduction is driven more by patrigenes.  The success of these predictions suggests that intragenomic conflict may occur in many contexts where matrigenes and patrigenes have different relatednesses to affected kin.

overall_design: Testing the kinship theory of intragenomic conflict in honey bees

first: Christina

last: Grozinger


title: Loss of motoneuron-specific microRNA-218 causes systemic neuromuscular failure [RNA-seq]

overall_design: Examination of mRNA expression in spinal progenitor, glial, and neuronal subpopulations.


title: EP400 Deposits H3.3 into Promoters and Enhancers During Gene Activation

city: Los Angeles

first: Siavash

last: Kurdistani

address: 615 Charles E Young Dr South

department: Biological Chemistry

institute: UCLA

summary:
Purpose: We wanted to know how histone variants H3.3 and H2AZ are deposited into genes and enhancers during gene activation
Methods: U2OS cells were harvested at 70% confluency with formaldehyde crosslinking for ChIP-seq without crosslinking for RNA-seq. ChIP DNA were purified through standard chromatin immunoprecipitation using an Pol II, MED26, EP400, H3.3, H2AZ, H3k4me1 and K3K18ac antibodies. Libraries were prepared with a KAPA LTP kit and sequenced using the Illumina HiSeq 2000 platform. Total RNA was extracted with Trizol, digested with DNaseI and further purified by acid phenol. Libraries were prepared with Illumina TruSeq RNA Sample Prep Kits v2 and were sequenced on Illumina HiSeq 2000.
Conclusion: Our biochemical and genomics (ChIP-Seq and  mRNA-Seq) data show that EP400 contributes to H3.3 deposition in significantly with less of an effect on H2AZ in both genes and enhancers

overall_design: Enrichment of EP400, Pol II, MED26, Histone varinat H3.3 and H2AZ, and enhancers marks H3K4me1 and H3K18ac in either Mock and/or EP400 knockdown conditions on chromatin were generated  by ChIP-Seq. mRNA profiles under Mock siRNA or EP400siRNA  were generated by deep sequencing, using Illumina HiSeq 2000.

first: Suman

last: Pradhan

first: Michael

last: Carey


title: Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase

first: Jared

last: Peace

state: CA - California

address: 1050 Childs Way RRI 203

department: Molecular and Computational Biology

institute: University of Southern California

type: Genome binding/occupancy profiling by array


title: A serum microRNA panel as potential biomarkers for hepatocellular carcinoma related with hepatitis B virus

city: Zhenjiang

first: youwen

last: tan

address: Danjianmen 300

institute: the third hospital of zhenjiang affiliated Jiangsu University

summary: Background: The identification of new high sensitivity and specificity markers for HCC are essential. We aimed to identify serum microRNAs for diagnosing hepatitis B virus (HBV) –related HCC.  Methods: Serum microRNA expression was investigated with four cohorts including 667 participants (261 HCC patients ,233 cirrhosi patients and 173 healthy controls), recruited between August 2010 and June 2013. First, An initial screening of miRNA expression by Illumina sequencing was performed using serum samples pooled from HCC patients and controls,respectively. Quantitative reverse-transcriptase polymerase chain reaction assay was then applied to evaluate the expression of selected microRNAs. A logistic regression model was constructed using a training cohort (n=357) and then validated using a cohort(n=241). The area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy.  Results: , We identified 8 miRNAs(hsa-miR-206, hsa-miR-141-3p, hsa-miR-433-3p, hsa-miR-1228-5p, hsa-miR-199a-5p, hsa-miR-122-5p, hsa-miR-192-5p and hsa-miR-26a-5p.)  formed a miRNA panel that provided a high diagnostic accuracy of HCC (AUC=0.887 and 0.879 for training and validation data set, respectively). The microRNA panel can also differentiate HCC from healthy (AUC =0.894) and cirrhosis (AUC = 0.892), respectively.  Conclusions:We found a serum microRNAs panel that has considerable clinical value in diagnosing HCC.

overall_design: 9 serum samples pooled from 3 healthy control donors and 3 HCC patients, 3 cirrhosi patients treated at The First Affiliated Hospital of Soochow University were subjected to Illumina HiSeq 2000 deep sequencing to identify the miRNAs that were significantly differentially expressed.

first: Youwen

last: Tan


title: CFTR is a tumor suppressor gene in murine and human intestinal cancer [RNA-seq]

city: Minneapolis

first: Ying

last: Zhang

address: 117 Pleasant St S.E.

department: Minnesota Supercomputing Institute

institute: University of Minnesota

summary: Analysis of the cystic fibrosis gene Cftr in the colon and small intestine of Cftr-deficient murine model. The hypothesis was loss of Cftr altered expression of genes important in intestinal homeostasis and oncogenic signaling pathways. The results identified potential roles of Cftr in up- or down-regulating major gene clusters that belong to groups of immune response, ion channel, intestinal stem cell and other growth regulators.

overall_design:
The experiments were designed to analyze the role of Cftr-deficiency in tumorigenesis. The goal of this study was to identify genes and pathways associated with Cftr-deficiency in Apc wildtype and ApcMin mice.  Total RNAs were isolated from mice, and subjected to deep sequencing, in duplicates, using Illumina HiSeq 2500. Samples that were sequenced in the same batch were analyzed in pair-wise using Tophat-Cuffdiff pipeline as outlined in Nature Protocol from Trapnell C. et al, 2012. The results indicated that Cftr-deficiency overlapped with genes and pathways involved in immune and inflammatory signaling, stem cell regulation, and Wnt/beta catenin signaling.
Total RNA was isolated from multiple colon tumors and multiple small intestine tumors from Apc wildtype Cftr-deficient mice, ApcMin Cftr-deficient mice, and ApcMin Cftr wildtype mice. Total RNA was also obtained from Apc wildtype normal colon (epithelial cells) and normal duodenum (whole duodenum minus villi) from three Cftr wildtype and three Cftr-deficient mice. RNA Seq was then conducted on all samples with at least two replicates for each biological sample.
Please note that 1) The 23 mice were processed in several batches, and two sequencing runs were carried out at two different dates.  To control for the batch effect of sequencing, some samples were included in both runs (run1 and run2).
2) To reach the desired sequencing depth and to keep loading balance, each sample was split into halves, and sequenced on two lanes (L007 and L008 for run1, L006 and L007 for run2).
therefore, for 11 samples, there are 4 technical replicates, including the 2-batches and 2-lane sequencing method.  For the remaining 12 samples, there are 2 technical replicates, referring to the 2-lane sequencing.
3) some of the mice are heterozygous mutant of CFTR gene (CFTRhet), named as "CFTR knockdown".

first: B

last: Than

first: J

middle: F

last: Linnekamp

first: T

last: Starr

first: D

last: Largaespada

first: A

last: Rod

first: Y

first: V

last: Bruner

last: Abrahante

last: Schumann

last: Luczak

last: Niemczyk

first: M

last: O’Sullivan

middle: P

last: Medema

first: R

middle: J

last: Fijneman

first: G

last: Meijer

first: E

last: Van den Broek

first: C

last: Hodges

first: P

last: Scott

first: L

last: Vermeulen

last: Cormier


title: Models of Human Core Transcriptional Regulatory Circuitries

city: Cambridge

first: Richard

last: Young

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: A small set of core transcription factors (TFs) dominates control of the gene expression program in embryonic stem cells and other well-studied cellular models. These core TFs collectively regulate their own gene expression, thus forming an interconnected autoregulatory loop that can be considered the core transcriptional regulatory circuitry (CRC) for that cell type. There is limited knowledge of core TFs, and thus models of core regulatory circuitry, for most cell types. We recently discovered that genes encoding known core TFs forming CRCs are driven by super-enhancers, which provides an opportunity to systematically predict CRCs in poorly studied cell types through super-enhancer mapping. Here we use super-enhancer maps to generate CRC models for 75 human cell and tissue types. These core circuitry models should prove valuable for further investigating cell type-specific transcriptional regulation in healthy and diseased cells.

overall_design: ChIP-Seq for RUNX1 and GATA3 in Jurkat T cell acute lymphoblastic leukemia cells.

first: Violaine

last: Saint-Andre

first: Alexander

last: Federation

first: Charles

middle: Y

last: Lin

first: Brian

last: Abraham

first: Jessica

last: Reddy

first: Tong

middle: I

last: Lee

first: James

last: Bradner


title: MLL-rearranged acute lymphoblastic leukemias upregulate BCL-2 through H3K79 methylation and are highly sensitive to the BCL-2 specific antagonist ABT-199

status: Public on Dec 17 2015

city: Oxford

first: Thomas

last: Milne

address: John Radcliffe Hospital

department: Molecular Haematology Unit

country: United Kingdom

institute: Weatherall Institute of Molecular Medicine

summary: We present ChIP-seq in the t(4;11) cell line SEM for different components of the MLL-AF4 complex as well as other chromatin proteins

overall_design: Examination of 5 histone marks, MLL, AF4, ENL and CFP1 in t(4;11) cells

first: Jon

last: Kerry


title: H3K36me3 is significantly more enriched at Set1-affected loci than at MLL1/2-affected loci in planarian stem cells

city: Happy Valley

first: Alex

last: Chitsazan

state: OR

address: 2720 S Moody Ave

institute: Oregon Health and Science University

summary: Histone H3 lysine 4 trimethylation (H3K4me3) is known to correlate with both active and poised genomic loci, yet many questions remain regarding its functional roles in vivo. We identify functional genomic targets of two H3K4 methyltransferases, Set1 and MLL1/2, in both the stem cells (X1) and differentiated tissue (Xins) of the planarian flatworm Schmidtea mediterranea. We show that, despite their common substrate, these enzymes target distinct genomic loci in vivo, which are distinguishable by the footprint each enzyme leaves on the chromatin template, i.e., the breadth of the H3K4me3 peak. Whereas Set1 targets are broadly associated with the maintenance of the stem cell population, MLL1/2 targets are specifically enriched for genes involved in ciliogenesis. These data not only confirm that chromatin regulation is fundamental to planarian stem cell function, but also provide evidence for post-embryonic functional specificity of H3K4me3 methyltransferases in vivo. To test this, we looked at the H3K4me3 chromatin profile in WW, X1 and Xins cell types upon knockdown of set1, mll1/2, as well as WT.

overall_design:
Examine binding of H3K36me3 in WT in Planarian. The experiment performed yielded a total of 2 samples.
These samples validate the main ChIP samples.

first: Elizabeth

middle: M

last: Duncan

middle: D

first: Chris

last: Seidel

first: Alejandro

last: Sanchez Alvarado


title: High-throughput sequencing of small RNAs (18-30 nt) from wild-type and diabetic mouse liver

city: Shanghai

first: Qiwei

last: Zhai

address: 294 Taiyuan Road

institute: Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

summary: Small RNA profiles in the liver of 9-week old wild type and diabetic db/db mice were measured to detect the differentially expressed small RNAs.

overall_design: Small RNA profiles of 9-week old wild type (WT) and diabetic db/db mice were generated by deep sequencing using Illumina Genome Analyzer.

first: Fang

last: Zhang

first: Haibin

last: Wei


title: Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase (BrdU)

first: Jared

last: Peace

summary: The S. cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering.  As stimulators of early origin activation, we hypothesized that Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide.  Existing methods, however, were not well suited to quantitative, genome-wide measurements of origin firing between strains and conditions.  To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advance the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase.  The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation.  The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing.  These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase.

overall_design: 5 total experiments with replicates

first: Sandra

middle: K

last: Villwock

first: John

middle: L

last: Zeytounian

first: Yan

last: Gan

first: Oscar

last: Aparicio


title: A Dicer-Independent Route for Biogenesis of siRNAs that Direct DNA Methylation in Arabidopsis

city: Beiing

first: Yijun

last: Qi

address: NO.1 Qinghuayuan

department: School of Life Sciences

institute: Tsinghua University

summary: DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. Twenty-four-nt siRNAs are known to be processed from double-stranded RNAs by Dicer-like 3 (DCL3) and loaded into the effector Argonaute 4 (AGO4). Here we report a distinct class of siRNAs independent of DCLs (sidRNAs). sidRNAs are present as ladders of ~20 to 60nt in length, often having same 5' ends but differing in 3' ends by 1-nt steps. We further show that sidRNAs are associated with AGO4 and capable of directing DNA methylation. Finally we show that sidRNA production depends on AGO4 and distributive 3'-5' exonucleases. Our findings suggest an alternative route for siRNA biogenesis: precursor transcripts are bound by AGO4 and subsequently subjected to 3'-5' exonucleolytic trimming for maturation. We propose that sidRNAs generated through this route are the initial triggers of de novo DNA methylation.

overall_design: Small RNAs were profiled in Arabidopsis wild type (Col-0), dcl1/2/3/4 and other mutants by Illumina high-throughput sequencing, to identify siRNAs independent of DCLs (sidRNAs) and dissect their biogenesis pathway. DNA methylation was profiled in various mutants by whole-genome bisulfite sequencing to establish a role for sidRNAs in DNA methylation

first: Ruiqiang

last: Ye

first: Zulong

last: Chen

first: Bi

last: Lian

first: Ning

last: Xia


title: Set1 and MLL1/2 target distinct sets of functionally different genomic loci in vivo: An in depth ChIP-seq analysis of H3K4me3 targets in vivo and upon Set1 and mll1 knockdown

first: Alex

last: Chitsazan

overall_design: Examine differential binding of H3K4me3 in different experimental conditions. The experiment performed yielded a total of 58 samples.


title: Genetic and Epigenetic Variation, but Not Diet, Shape the Sperm Methylome

city: Worcester

first: Alper

last: Kucukural

address: 364 Plantation Street

department: Program in Molecular Medicine

institute: UMass Medical School

summary: The diet consumed by fathers prior to procreation impacts metabolic phenotypes in their offspring, but the mechanisms underlying such intergenerational information transfer remain obscure. Here, we carried out extensive analysis of cytosine methylation patterns in murine sperm, generating whole genome methylation maps for 4 pools of sperm samples and for 12 individual sperm samples, as well as 61 genome-scale reduced-representation bisulfite sequencing (RRBS) methylation maps, using samples obtained from male mice consuming various diets. We found that epivariation, either stochastic or due to unknown demographic or environmental factors, was a far stronger contributor to the sperm methylome than was the diet consumed. Variation in cytosine methylation was particularly dramatic over tandem repeat families, including ribosomal DNA (rDNA) repeats, and rDNA methylation levels were heritable from one generation to the next. However, rDNA methylation was strongly correlated with genetic variation in rDNA copy number, and analysis of hundreds of sperm samples revealed no consistent effect of diet on rDNA copy number or methylation level in sperm, indicating that paternal diet exerts an rDNA methylation-independent effect on offspring gene expression. These results reveal loci of genetic and epigenetic lability in the mammalian genome, but strongly argue against a direct mechanistic role for sperm cytosine methylation in dietary reprogramming of offspring metabolism.

overall_design:
4 Pooled WGBS libraries are analyzed. Each pool contains DNA from 7-8 mice.

Methylation call data could not be located. Processed data not available for this study.

first: Jeremy

last: Shea

first: Ryan

last: Serra

first: Benjamin

middle: R

last: Carone

first: Hennady

last: Shulha

first: Michael

last: Ziller

first: Marcus

last: Vallaster

first: Hongcang

last: Gu

first: Andrew

last: Tapper

first: Paul

last: Gardner

first: Alexander

last: Meissner

first: Manuel

last: Garber

first: Oliver

last: Rando


title: Set1 and MLL1/2 target distinct sets of functionally different genomic loci in vivo: An in depth RNA-seq analysis of H3K4me3 targets in vivo and upon Set1 and mll1 knockdown

summary: This study identifies expression differences upon knockdown of two H3K4 methyltransferases, Set1 and MLL1/2, in both the stem cells (X1) and whole worm (WW) tissue of the planarian flatworm Schmidtea mediterranea. To test this, we looked at the RNA-seq expression profile in WW and X1 tissue types upon RNAi knockdown of set1, mll1/2 and compared to a non-targeted RNAi control, unc22.

overall_design: Examine gene expression profiles of adult flatworm’s X1 and WW tissue type upon knockdown of mll1/2 and set1. These were then compared to a non-targeted RNAi control, unc22.

first: Elizabeth

last: Duncan

first: Chris

last: Seidel

first: Alejandro

last: Sanchez Alvarado


title: Identification and characterization of MCF01A circular RNAs [ChIP-seq]

city: Rehovot

first: Yehoshua

last: Enuka

address: Herzl 234

department: Biological Regulation

institute: Weizmann Institute of Science

summary: Circular RNAs (circRNAs) are widespread circular forms of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of both coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and micro-RNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms only. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes.

overall_design: Histone 3 Lysine 27 Acetylation – 2 replicates


title: Identification and characterization of MCF01A circular RNAs [RNA-seq]

overall_design: Detection of circRNAs from RNA-Seq – triplicate


title: Identification and characterization of MCF01A circular RNAs

overall_design: Refer to individual Series


title: NSD3-short is an adaptor protein that couples BRD4 to the CHD8 chromatin remodeler

city: Cold Spring Harbor

first: Christopher

middle: R.

last: Vakoc

state: New York

address: One Bungtown Road

institute: Cold Spring Harbor Lab


title: Gene expression alterations in SoxC knockdown sea urchin embryos

status: Public on Dec 16 2015

city: Bethesda

first: Robert

middle: C

last: Angerer

address: 30 Convent Drive

institute: NIDCR

summary: Transcription factor SoxC is required for all neural development in purple sea urchin S. purpuratus embryos. To begin to develop a gene regulatory network for neural development, we used RNA-Seq to compare transcript populations in SoxC knockdown and control embryos.

overall_design: SoxC function was knocked down by morpholino oligo injection. RNA from about 1000 embryos were collected for both control and knockdown samples.

first: Zheng

last: Wei

first: Lynne


title: CFTR is a tumor suppressor gene in murine and human intestinal cancer

first: Ying

last: Zhang

state: MN


title: NSD3-short is an adaptor protein that couples BRD4 to the CHD8 chromatin remodeler (RNA-Seq)

summary: The bromodomain and extraterminal (BET) protein BRD4 is a therapeutic target in acute myeloid leukemia (AML). Here, we demonstrate that the AML maintenance function of BRD4 requires its interaction with NSD3, which belongs to a subfamily of H3K36 methyltransferases. Unexpectedly, AML cells were found to only require a short isoform of NSD3 that lacks the methyltransferase domain. We show that NSD3-short is an adaptor protein that sustains leukemia by linking BRD4 to the CHD8 chromatin remodeler, by using a PWWP chromatin reader module, and by employing an acidic transactivation domain. Genetic targeting of NSD3 or CHD8 mimics the phenotypic and transcriptional effects of BRD4 inhibition. Furthermore, BRD4, NSD3, and CHD8 colocalize across the AML genome, and each is released from super-enhancer regions upon chemical inhibition of BET bromodomains. These findings suggest that BET inhibitors exert therapeutic effects in leukemia by evicting BRD4-NSD3-CHD8 complexes from chromatin to suppress transcription.

overall_design: PolyA+ (illumine TruSeq)/not-so-random (NSR) primers selected RNA-Seq for shRNA/sgRNA-expressing MLL-AF9 transformed acute myeloid leukemia cells (RN2).

first: Chen

last: Shen


title: Combinatorial Control of RBP-jk and EBF1 Chromosome Occupancy induced by viral co-activator EBNA2 [ChIP-seq]

city: Philadelphia

first: Priyankara

last: Wickramasinghe

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: Gene expression programs depend on sequence-specific DNA binding transcription factors, but the mechanisms that control the selective binding of these factors in a chromosomal and genomic context remain enigmatic.   Here, we show that two master regulators of B-cell fate, namely EBF1 and RBP-jk, show variable genome-wide chromosome distribution in two related B-lymphocyte lines carrying different forms of Epstein-Barr Virus (EBV) latency.  The latency-type specific EBV-encoded EBNA2 colocalized with RBP-jk and EBF1 at induced binding sites.  Colocalization of EBF1, RBP-jk, and EBNA2 correlated with transcriptional activation.  Conditional expression or repression of EBNA2 lead to a rapid alteration in RBP-jk and EBF1 binding.  Biochemical and shRNA depletion studies provide evidence for cooperative assembly at co-occupied sites.  These findings reveal that non-DNA binding cofactors can facilitate combinatorial interactions to induce new patterns of transcription factor occupancy and gene programming

overall_design: Examination of EBNA2/EBF1/EBP-jk binding in MutuI and LCL cell lines

last: Lieberman

first: Fang

last: Lu