title: A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture

city: Davis

first: Henriette

last: O'Geen

address: 1 Shields Ave

department: Genome Center

institute: Uc Davis

summary: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of their target site recognition. Most previous studies have examined predicted off-target binding sites that differ from the perfect target site by one to four mismatches, which represent only a subset of genomic regions. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites. For two guide RNAs targeting the murine Snurf gene promoter, we observed very high binding specificity at the intended target site while off-target binding was observed at 2- to 6-fold lower intensities. We also identified significant gRNA-independent off-target binding. Interestingly, we found that these regions are highly enriched in the PAM site, a sequence required for target site recognition by CRISPR. To determine the relationship between Cas9 binding and endonuclease activity, we used targeted sequence capture as a high-throughput approach to survey a large number of the potential off-target sites identified by ChIP-seq or computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results demonstrate that even a simple configuration of a Cas9:gRNA nuclease can support very specific DNA cleavage activity and that most interactions between the CRISPR nuclease complex and genomic PAM sites do not lead to DNA cleavage.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
ChIP-seq using dCas9 to determine genome-wide binding of CRISPR/Cas9

noED: Cas9 doublemutant protein without an effector domain
KRAB: Cas9 doublemutant protein fused to the KRAB repressor domain
S1 gRNA: guide RNA targeting GCTCCCTACGCATGCGTCCC(AGG) in the mouse genome
S2 gRNA: guide RNA targeting AATGGCTCAGGTTTGTCGCG(CGG) in the mouse genome
VEGFA TS3 gRNA: guide RNA targeting GGTGAGTGAGTGTGTGCGTG(TGG) in the human genome

first: Isabelle

last: Henry

first: David

last: Segal


title: Ectoderm specification of H1 human embryonic stem cells

status: Public on Mar 05 2015

city: New Haven

first: Valerie

last: Horsley

address: 260 Whitney

country: USA

institute: Yale University

type: Expression profiling by high throughput sequencing

summary: In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ–secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.

overall_design: 6 samples were analyzed, 3 replicates of ETOH treated H1 HESCs and 3 replicates of DAPT treated H1 HESCs

first: Ana

last: Tadeu

first: Samantha

last: Lin

first: Lin

last: Hou

first: Lisa

last: Chung

first: Mei

last: Zhong

first: Hongyu

last: Zhao


title: Role of RfaH in Yersinia enterocolitica O:3

status: Public on Mar 04 2015

city: Helsinki

first: Päivi

last: Saavalainen

address: Haartmaninkatu 8

department: TRIMM

country: Finland

institute: University of Helsinki

summary: To assess the effects of rfaH mutation on gene expression, RNA sequencing was carried out using the RNA extracted from rfaH mutant and wild type bacteria cultivated at both 22˚C and 37˚C. Additionally, to differentiate between genes affected directly by the rfaH mutation and those affected by the O-antigen negative phenotype, the Y. enterocolitica O:3 strain YeO3-R1 missing the O-antigen was included in the RNA-sequencing study. At RT-grown bacteria altogether 77 (45 up- and 32 down-regulated) differentially expressed genes were identified. At 37ºC, on the other hand, 44 genes were differentially expressed; 14 genes up- and 30 genes down-regulated. The RNA-sequencing data of YeO3-R1 was available only for bacteria grown at 22˚C and the comparison revealed that 22 of the 102 genes were similarly differentially expressed both in rfaH and YeO3-R1 mutants.  To detect genes regulated directly or indirectly by Hfq, deep RNA-seq was performed with RNA isolated from Y. enterocolitica wild-type strain 6471/76 and the YeO3-hfq::Km strain. Total RNA was isolated from two biological replicas of bacteria grown in LB to logarithmic phase (OD600 = 0.6) at two different temperatures, RT and 37°C. knocking out of Hfq affected the transcription of 346 genes at RT and 541 genes at 37°C, i.e., ca. 8 and 12.5 % of the Y. enterocolitica genes, respectively. At RT, of the 346 genes 216 genes were down- and 132 up-regulated, and at 37°C, of the 541 genes, 96 were down- and 445 up-regulated. A total of 95 genes were differentially expressed both at RT and 37°C; 27 showed down- and 59 up-regulated. For 9 genes opposite changes took place depending on the growth temperature.

overall_design: Bacterial RNA profiles of wild type, rfaH- and hfq- strains were generated using deep sequencing, in duplicates, under two growth temperatures (22°C and 37°C). The RNA-sequencing data of YeO3-R1 was generated only from bacteria grown at 22˚C.

first: Katarzyna

last: Leskinen

first: Pirkko

last: Mattila


title: Linking the aryl hydrocarbon receptor with altered DNA methylation patterns and developmentally induced aberrant antiviral T cell responses [RNA-Seq]

status: Public on Mar 16 2015

city: Rochester

first: B Paige

last: Lawrence

state: NY

address: 575 Elmwood Ave.

department: Environmental Medicine

institute: University of Rochester Medical Center

summary: Successfully fighting infection requires a properly tuned immune system. Recent epidemiological studies link exposure to pollutants that bind the aryl hydrocarbon receptor (AHR) during development with poorer immune responses later in life. Yet, how developmental triggering of AHR durably alters immune cell function remains unknown. Using a mouse model, we show that developmental activation of AHR leads to long-lasting reduction in the response of CD8+ T cells during influenza virus infection, cells critical for resolving primary infection. Combining genome-wide approaches, we demonstrate that developmental activation alters DNA methylation and gene expression patterns in isolated CD8+ T cells prior to and during infection. Altered transcriptional profiles in CD8+ T cells from developmentally exposed mice reflect changes in pathways involved in proliferation and immunoregulation, with an overall pattern that bears hallmarks of T cell exhaustion. Developmental exposure also changed DNA methylation across the genome, but differences were most pronounced following infection, where we observed inverse correlation between promoter methylation and gene expression. This points to altered regulation of DNA methylation as one mechanism by which AHR causes durable changes in T cell function. Discovering that distinct gene sets and pathways were differentially changed in developmentally exposed mice prior to and after infection further reveals that the process of CD8+ T cell activation is rendered fundamentally different by early life AHR signaling. These findings reveal a novel role for AHR in the developing immune system: regulating DNA methylation and gene expression as immune cells respond to viral infection later in life.

overall_design: In this study, two biological replicates were collected for each of four treatment groups: developmental exposure to vehicle control (Veh) and naïve, developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and naïve, developmental exposure to Veh and infected, developmental exposure to TCDD and infected. For each sample, both RNA-seq and methylated DNA immunoprecipitation (MeDIP)-seq were performed.

first: Bethany

last: Winans

first: Anusha

last: Nagari

first: Minho

last: Chae

first: Chia-I

last: Ko

first: Alvaro

last: Puga

first: W

middle: L

last: Kraus

first: B

middle: P


title: Unraveling determinants of transcription factor binding outside the core binding site

city: Rehovot,

first: Michal

last: Levo

address: Herzl Street 234

country: Israel

institute: Weizmann Institute of Science

summary: Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences now serving and promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences, and allows the characterization of TF binding determinants within and outside of core binding sites.

overall_design: Binding experiments were carried out with one of two input libraries; Lib1 (also referred to as “main lib”) includes designed sequence variants of length 150bp, and Lib2 (also referred to as “constant flanks lib”) includes designed sequence variants of length 200bp. The sequences of the variants of interest can be found in the second column of the processed data file (BunDLE-seq_data, see 3 excel sheets). EXP1, EXP2 and EXP3 were carried out using Lib1, and EXP4 was carried out using Lib2.  Binding experiments were carried out with different proteins (in different concentrations) – DNA was extracted from bound bands that were formed on the gel and amplified with a primer with a unique 5bp upstream tail specifying the identity of the originating band (also referred to as “band barcode”). Data from the following bands was used in the analysis:  EXP1_band1, no-protein band – with band barcode “AACGA”, EXP1_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “TCGTA” , EXP1_band3, histones bound band – with band barcode “AGATA”, EXP2_band1, no-protein band – with band barcode “AACGA”, EXP2_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “AGATA”, EXP2_band2, single Gcn4 (conc 1:7) bound band – with band barcode “CACTT”, EXP2_band2, single Gcn4 (conc 1:6) bound band – with band barcode “TCGTA”, EXP2_band2, single Gcn4 (conc 1:5) bound band – with band barcode “CATCA”, EXP2_band2, single Gcn4 (conc 1:4) bound band – with band barcode “TATTA”, EXP2_band2, single Gcn4 (conc 1:3) bound band – with band barcode “AGTCA”, EXP2_band2, single Gcn4 (conc 1:2) bound band – with band barcode “ACGAA”, EXP2_band2, single Gcn4 (conc 1:1) bound band – with band barcode “ACAGA”, EXP2_band2, two Gcn4 (conc 1:1) bound band – barcoded with “GTAGA”, EXP3_band1, no-protein band – with band barcode “GCATA”, EXP3_band2, single Gal4 (conc 1:1) bound band – with band barcode “GATGT”, EXP3_band3, single Gal4 (conc 3:1) bound band – with band barcode “GTTCA”, EXP3_band4, two Gal4 (conc 3:1) bound band – with band barcode “CTCAA”, EXP4_band1, no-protein band – with band barcode “GAGTA”, EXP4_band2, single Gcn4 (conc 1:3) bound band –  “TACCA”, EXP4_band3, no-protein band – with band barcode “TCGTT”, EXP4_band3, single Gal4 (conc 3:1) bound band – with band barcode “ACATC”, *biological replicates

first: Einat

last: Zalckvar

first: Eilon

last: Sharon

first: Ana-Carolina

last: Dantas Machado

first: Yael

last: Kalma

first: Maya

last: Lotam-Pompan

first: Adina

last: Weinberger

first: Zohar

last: Yakhini

first: Remo

last: Rohs

first: Eran

last: Segal


title: DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes

status: Public on Mar 03 2015

city: Ann Arbor

first: Mats

last: Ljungman

state: Michigan

address: NCRC, B520 Room 1346 2800 Plymouth Rd.

institute: University of Michigan

summary: The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5’-3’ direction with slow recovery and TC-NER at the 3’ end of long genes. RNA synthesis resumed fully at the 3’-end of genes after a 24-hour recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases.

overall_design: This study is composed of three identical experiments run in three different cell lines. For each experiment, there is one control (mock irradiated cells) and four test samples (0h, 2h, 6h and 24h after UV 10J irradiation).

first: Leonardo

middle: C

last: Andrade-Lima

first: Artur

last: Veloso

first: Michelle

middle: T

last: Paulsen

first: Carlos

middle: F

last: Menck


title: To identify ATFS-1 target genes during mitochondrial stress

city: New York

first: Jeffrey

last: Zhao

address: 1275 York Avenue

department: Genomics Core

institute: Memorial Sloan Kettering Cancer Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: To better understand genome coordination and OXPHOS recovery during mitochondrial dysfunction, we examined ATFS-1, a transcription factor that regulates mitochondria-to nuclear communication during the mitochondrial UPR, via ChIP-sequencing.

overall_design: Wildtype worms treated spg-7(RNAi) are analyzed in the presence and absence of ATFS-1 antibody to identify ATFS-1 targets. Individual samples were analyzed. Wildtype worms treated spg-7(RNAi) in the absence of antibody is used as a control.

first: Amrita

middle: M

last: Nargund

first: Cole

last: Haynes


city: Chicago

first: Alex

last: Ruthenburg

address: 920 E. 58th Street

institute: University of Chicago

summary: Biochemical fractionation of HEK293 nuclei and RNA-seq of chromatin-associated and soluble-nuclear RNA.

overall_design: Nuclei from three biological replicates were isolated by detergent lysis, fractionated, then three chromatin and three soluble RNA samples were converted to cDNA using Illumina TruSeq stranded protocol, and sequenced on Illumina HiSeq2000

first: Michael

middle: S

last: Werner

middle: J


title: A genetic circuitry linking Id-proteins (Id2 and Id3) and the AKT-FOXO-mTORC1 axis to suppress innate-variant TFH cell development, maintain T cell quiescence and prevent lymphomagenesis.

city: La Jolla

first: Kazuko

last: Miyazaki

state: CA

address: 9500 Gilman Drive

department: Division of Biological Sciences

institute: University of California, San Diego

summary: It is now well established that the E- and Id-protein axis regulates multiple steps in lymphocyte development. However, it remains unknown as to how E- and Id-proteins mechanistically enforce and maintain the naïve T cell fate. Here we show that Id2 and Id3 suppressed the development and expansion of innate-variant TFH cells. Innate-variant TFH cells required MHC Class I-like signalling and were associated with germinal center B cell development. We found that Id2 and Id3 induced Foxo1 and Foxp1 expression to antagonize the activation of TFH transcription signature.  We show that Id2 and Id3 acted upstream of the Hif1a/Foxo/AKT/mTORC1 pathway as well as the c-myc/p19Arf module to control cellular expansion and activation. We found that mice depleted for Id2 and Id3 expression developed colitis and αβ T cell lymphomas. Lymphomas depleted for Id2 and Id3 expression displayed elevated levels of c-myc whereas p19Arf abundance declined. Transcription signatures of Id2- and Id3-depleted lymphomas revealed similarities with genetic deficiencies associated with Burkitt lymphoma. We propose that in response to antigen receptor and/or cytokine signaling the E-Id protein axis modulates the activities of the PI3K-AKT-mTORC1-Hifa and c-myc/p19Arf pathways to control cellular expansion and homeostatic proliferation.

overall_design: RNA-seq data of 5 of wild type CD4SP cells, 3 of wild type Tfh cells, 3 of Id3-/- CD4SP cells, 3 of Id2-/-Id3-/-(dKO) CD4SP cells, and 6 of Id2-/-Id3-/- lymphoma cells.


title: Direct but No Transgenerational Effects of Decitabine and Vorinostat on Male Fertility

city: Heidelberg

first: Christian

last: Rohde

address: Im Neuenheimer Feld 410

country: Germany

institute: Heidelberg University

type: Methylation profiling by high throughput sequencing

summary: Epigenetic drugs actively altering the epigenome of tumours have been developed for use in anti-cancer therapies. However, these drugs could potentially also affect the DNA methylome of spermatozoa that seems to be essential for male fertility. Here, we analysed possible direct and transgenerational effects of the epigenetic drug decitabine on the DNA methylome of spermatozoa. Our analysis revealed the absence of gross differences between the spermatozoal methylome of decitabine treated and untreated animals as well as between their F3 generations. Interestingly, the methylomes of spermatozoa from the selected mice of the F3-generations were also highly similar to the analysed parental samples.

overall_design: We studied genome-wide DNA methylation changes in decitabine treated mice and their F3-generation. For this, we treated male C57BL/6 mice (seven weeks old) for seven weeks (intraperitoneal, three times per week) with the DNMT inhibitor decitabine (5-aza-2'-deoxycytidine, 0.1 µg/g, n = 17) or with vehicle (7.5 % dimethyl sulfoxide in phosphate buffered saline, n = 16). The dose of decitabine per treatment was chosen according to the clinical application regimens in humans and refer to previous mice studies using this drug (Kelly et al, 2003; Oakes et al, 2007). For the investigation of transgenerational effects, 10 treated and untreated male mice were mated with four untreated female C57BL/6 mice each. The offspring was considered as F1-generation. The F2- and F3-generations were obtained by an identical mating scheme. Thus, only the P-generation males were treated with decitabine or control vehicle, the F1-, F2- and F3-generations were not subjected to any treatment. During this study, all mice were housed at 24 °C on a 12-h light, 12-h dark cycle and provided with food and tap-water ad libitum.

first: Ruth

last: Kläver

first: Carsten

last: Müller-Tidow


title: Transcriptional control in Candidatus Pelagibacter ubique and other marine bacterial taxa

city: Lewes

first: Matthew

last: Cottrell

address: 700 Pilottown Rd

institute: University of Delaware

summary: Bacteria respond to stimuli in the environment using transcriptional control, but this may not be the case for most marine bacteria having small, streamlined genomes.  Candidatus Pelagibacter ubique, a cultivated representative of the SAR11 clade, which is the most abundant clade in the oceans 4, has a small, streamlined genome and possesses an unusually small number of transcriptional regulators.  This observation leads to the hypothesis that transcriptional control is low in Pelagibacter and limits its response to environmental conditions.  However, the extent of transcriptional control in Pelagibacter is unknown.  Here we show that transcriptional control is extremely low in Pelagibacter and another oligotroph (SAR92) compared to two marine copiotrophic bacterial taxa, Polaribacter MED152 and Ruegeria pomeroyi.  We found that ~0.1% of protein-encoding genes in Pelagibacter are under transcriptional control compared to >10% of genes in other marine bacteria.  Regardless of the growth condition, the same genes were highly expressed while most genes were always expressed at very low levels.  Quantitative RNA sequencing revealed that abundances of most Pelagibacter transcripts were <0.01 copies per cell whereas transcript abundances were 1 to 10 copies per cell in some other bacteria.  Our results demonstrate that Pelagibacter can change growth without shifts in transcript levels, suggesting that transcriptional control plays a minimal role in the adaptive strategy for one of the most successful organisms in the biosphere.

sample_organism:
Ruegeria pomeroyi DSS-3
Polaribacter sp. MED152
gamma proteobacterium HTCC2207
Candidatus Pelagibacter ubique HTCC1062

overall_design: Bacteria were grown in batch culture and sampled twice during the initial, rapid phase of exponential growth and twice during the phase of slower growth that followed.

first: David

last: Kirchman


title: The ß-importin KAP8 (Pse1/Kap121) is required for nuclear import of the cellulase transcriptional regulator XYR1, asexual sporulation and stress resistance in Trichoderma reesei

city: PARIS

first: sophie

last: lemoine

address: 46 rue d'Ulm

department: Biologie

country: France

institute: Ecole Normale Supérieure

summary: The ascomycete Trichoderma reesei is an industrial producer of cellulolytic and hemicellulolytic enzymes and also serves as a model for investigations on these enzymes and their genes. Most of them are obligatorily dependent on the Zn(II)Cys(VI) transcriptional activator XYR1. XYR1 is constitutively expressed at a low basal, but induced in the presence of cellulase- and hemicellulose inducers, and transported into the nucleus. The factors mediating its import and export across the nuclear pore complexes (karyopherins) are therefore expected to play a key role in its function. We identified 14 karyopherins in T. reesei, of which 8 were predicted to be involved nuclear protein import. Their systematic knock-out and testing for cellulase formation identified KAP8 (an orthologue of A. nidulans KapI, and Saccharomyces cerevisiae Kap121/Pse1p) to be essential for cellulase gene expression, and for the appearance of GFP-XYR1 in the nucleus. Transcriptomic analysis of Δkap8 and a retransformant under cellulase-inducing conditions revealed the downregulation of 64 Cazymes in the Δkap8 strain under inducing conditions, including all cellulases and hemicellulases known to be under XYR1 control, and of 12 transcription factors of which 2 were known to be associated with cellulase regulation (ACE3, CLR2). Together, these new observations underscore the role of nuclear transport of XYR1 in the regulation of cellulase and hemicellulose gene expression in T. reesei, and identify KAP8 as the major karyopherin involved in this process.

overall_design: Two strains were used: T. reesei ku70 (pyr4-), in which the kap8 (=kapI/Pse1/Kap121) reading frame has been replaced by the T. reesei pyr4 gene; and a kap8-retransformant of the same strain and two time points for each strain T0h and T3h. All samples are in tricplicate.

first: F

last: Bidard

first: S

last: Lemoine

first: MN

last: Rossignol

first: C

last: Kubicek


title: RNA Sequencing Analysis of Mouse Bone Marrow Derived Dendritic Cells Transcriptomes During Maturation

city: Shanghai

first: PIN

last: WANG

address: Xiangyin Road 800

country: China

institute: SMMU

summary:
Purpose:The purpose of this study is to detect activated or silenced genes during LPS-induced dendritic cell maturation. Gene expression differences between two samples could be found using transcriptome profiling (RNA-seq) analysis.
Methods:Mouse dendritic cells were generated from bone marrow cells in  RPMI-1640 medium with recombinant mouse GM-CSF and IL-4, mature DCs were obtained after LPS induced maturation. Immature DCs and mature DCs were sorted respectively based on maturation marker CD86 and Iab(MHCII) using flowcytrometer. DC mRNA profiles were generated by deep sequencing,using Illumina
Results: We mapped about 10 million sequence reads per sample to the mouse genome, identified 1,300 upregulated genes and 1,475 dow regulated genes during dendritic cell maturation.

overall_design: DC mRNA profiles immature and mature moouse BMDCs were generated by deep sequencing

first: Liu

last: Yiqi

first: Cao

last: Xuetao

first: Zhang

last: Qian


title: RNA-seq Analysis of Mineralizing IDG-SW3 Cultures with Mature Osteocyte Characteristics

city: San Antonio

first: stephen

middle: e

last: harris

state: TX

address: 7703 Floyd Curl Drive

department: Periodontics

institute: U. of Texas Health Sci Center at San Antonio

summary: We differentiated the murine IDG-SW3 cell line for 28 days until the cells displayed a mature osteocyte-like phenotype. Triplicate cultures of the IDG-SW3 cells were then treated with 50nM PTH (1-34) or vehicle control (PBS) for 24 hours. RNA was harvested from the cultures and used to perform RNA Seq analysis. We found that many genes previously shown to be markers of the osteocyte phenotype were strongly downregulated in response to PTH treatment. Furthermore, we found that genes known to inhibit cell motility were downregulated in response to PTH, whereas genes promoting motility were upregulated. This corresponds to the increased cell motility observed in PTH-treated IDG-SW3 cell cultures. Therefore, PTH induces a switch in mature IDG-SW3 cells from a osteocyte-like cell to a more motile phenotype.

overall_design: RNA expression profiles of control and PTH-treated 28 day differentiated IDG-SW3 cells.

first: Mathew

last: Prideaux

first: Lynda

last: Bonewald

first: Stephen

middle: E

last: Harris


title: Genome-wide analysis of histone modifications in human endometrial stromal cells [RNA-Seq]

city: Ube

first: Isao

last: Tamura

address: Minami-Kogushi 1-1-1

department: Department of Obstetrics and Gynecology

country: Japan

institute: Yamaguchi University Graduate School of Medicine

summary: Dramatic changes of gene expressions are known to occur in human endometrial stromal cells (ESC) during decidualization.  The changes in gene expression are associated with changes of chromatin structure, which are regulated by epigenetic mechanisms such as histone modifications.  Here, we investigated genome-wide changes in histone modifications and mRNA expressions associated with decidualization in human ESC using chromatin immunoprecipitation (ChIP) combined with next-generation sequencing.  ESC were incubated with estradiol and medroxyprogesterone acetate for 14 days to induce decidualization.  The ChIP-sequence data showed that induction of decidualization increased H3K27ac and H3K4me3 signals in many genomic regions but decreased in only a few regions.  Most (80%) of the H3K27ac-increased regions and half of the H3K4me3-increased regions were located in the distal promoter regions (more than 3 kb upstream or downstream of the transcription start site).  RNA-sequence showed that induction of decidualization up-regulated 881 genes, 223 of which had H3K27ac- or H3K4me3-increased regions in the proximal and distal promoter regions.  Induction of decidualization increased the mRNA levels of these genes more than it increased the mRNA levels of genes without H3K27ac- or H3K4me3-increased regions.  Pathway analysis revealed that up-regulated genes with the H3K27ac- or H3K4me3-increased regions were associated with insulin signaling.  These results show that histone modification statuses genome-widely change in human ESC by induction of decidualization.  The main changes of histone modifications are increases of H3K27ac and H3K4me3 in both the proximal and distal promoter regions, which are involved in the up-regulation of gene expression that occurs during decidualization.

overall_design: mRNA profiles of human endometrial stromal cells with and without EP inductions for 2 individuals. (EP induction: induction with estradiol (10-8 M) and medroxyprogesterone acetate (10-6 M))

first: Tamura

last: Isao

first: Sugino

last: Norihiro


title: Sequential centromere shift via de novo formation in maize

city: Wuhan

first: Handong

last: Su

state: Hubei

address: No.1,Shizishan Street · Hongshan District

institute: Huazhong Agricultural University

summary: The ability of centromeres to alternate between active and inactive states indicates significant epigenetic elements controlling centromere assembly and centromere function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In derivatives of TB-9Sb, we found a de novo centromere on chromosome telo-3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. This centromere is much smaller than normal ones but can be maintained through meiosis. The functional B centromere in progenitor telo2-2 is deleted from telo3-3 but some B-repeat sequences remain. The de novo centromere of telo3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on the chromosomes. One such de novo centromere contains only 200-kb CENH3 binding domain. This 200-kb centromere is located 3 Mb removed from the translocation breakpoint in a new location. The deleted B centromere in telo3-3 is activated in two derivatives. Our results suggest that de novo centromere formation is more common than previously thought and can persist on chromosomal fragments without a canonical centromere providing implications for karyotype evolution.

overall_design: ChIP-seq was carried out with anti-CENH3 antibodies using material from young leaves with control, telo3-3 and its derivate.

first: Fangpu

last: Han

first: James

middle: A

last: Birchler


title: Genome-wide analysis of histone modifications in human endometrial stromal cells [ChIP-Seq]

overall_design: Examination of 4 different histone modifications in human endometrial stromal cells with or without EP inductions for 2 individuals. (EP induction: induction with estradiol (10-8 M) and medroxyprogesterone acetate (10-6 M))


title: Nucleosome positioning in the IMR90 cell line

city: Basel

first: Dimos

last: Gaidatzis

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute

summary: Micrococcal nuclease (Mnase) treatment and sequencing of mononucleosomal DNA

overall_design: One single sample in the IMR90 fibroblast cell line

first: Lukas

last: Burger

first: Rabih

last: Murr

first: Anita

last: Lerch

first: Sophie

last: Dessus-Babus

first: Dirk

last: Schubeler

first: Michael

middle: B

last: Stadler


title: Genome-wide RING1B binding in Fbxl10 overexpressing 3T3-L1 cells at day 2 of differentiation

city: meguro-ku

first: Takeshi

last: Inagaki

state: Tokyo

address: 4-6-1 Komaba

institute: Univ of Tokyo

summary: We analyzed RING1B binding regions in 3T3-L1 cell lines transduced with the retroviral vector for V5-tagged Fbxl10 or its dF-box mutant.

overall_design: RING1B ChIP-seq in empty, Fbxl10-1, and dF-box mutant vector transduced 3T3-L1 preadipocytes, in duplicate, and V5-Fbxl10 ChIP-seq in Fbxl10 overexpressing 3T3-L1 cells


title: Gene expression profiling of wildtype vs. transgenic mice expressing the cardiac specific genetically encoded FRET biosensor Epac1-PLN

status: Public on Mar 02 2015

city: Goettingen

first: Gabriela

last: Salinas

state: Lower-Saxony

address: Kreuzbergring 57

department: Department of Pathology

institute: Universitaetsmedizin Goettingen

summary: 3’,5’-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger which regulates multiple physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors have been developed and used in cell lines or neonatal cardiomyocytes, it is unclear whether such biosensors can be successfully used in vivo, especially in the context of disease. Here, we generated the first transgenic mouse model expressing a targeted cAMP sensor (Epac1-PLN) and analyzed microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signaling in adult cardiomyocytes isolated from healthy mouse heart and from in vivo cardiac disease model. Finally, we used gene arrays to verify that the mRNA expression levels of the main players involved in cAMP signaling such as beta-ARs, G-protein, adenylyl cyclases, PKAs, PDEs, and Epac were not significantly changed between wildtype and transgenic Epac1-PLN expressing cardiomyocytes.

overall_design: To compare cardiac specific mRNA expression between wildtype and Epac1-PLN transgenic mice, cardiomyocyte RNA from total of 5 individual wildtype and 5 Epac1-PLN transgenic mice was isolated and submitted for the Illumina gene array analysis to the Göttingen transcriptome analysis laboratory (TAL).

first: Julia

middle: U

last: Sprenger

first: Viacheslav

middle: O

last: Nikolaev


title: Base-resolution methylation patterns accurately predict transcription factor bindings in vivo

city: Atlanta

first: Bing

last: Yao

state: GA

address: 615 Michael St. Rm 323

department: Human Genetics

institute: Emory University

summary: Detecting in vivo transcription factor (TF) binding is important for understanding gene regulatory circuitries. ChIP-seq is a powerful technique to empirically define TF binding in vivo. However, the multitude of distinct TFs makes genome-wide profiling for them all labor-intensive and costly. Algorithms for in silico prediction of TF binding have been developed, based mostly on histone modification or DNase I hypersensitivity data in conjunction with DNA motif and other genomic features. However, technical limitations of these methods prevent them from being applied broadly, especially in clinical settings. We conducted a comprehensive survey involving multiple cell lines, TFs, and methylation types and found that there are intimate relationships between TF binding and methylation level changes around the binding sites. Exploiting the connection between DNA methylation and TF binding, we proposed a novel supervised learning approach to predict TF-DNA interaction using data from base-resolution whole-genome methylation sequencing experiments. We devised beta-binomial models to characterize methylation data around TF binding sites and the background. Along with other static genomic features, we adopted a random forest framework to predict TF-DNA interaction. After conducting comprehensive tests, we saw that the proposed method accurately predicts TF binding and performs favorably versus competing methods.

overall_design: Examine Oct4 genome-wide binding in mouse embryonic stem cells (E14)


title: Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion

city: Dresden

first: Mareike

last: Albert

address: Fetscherstr. 105

department: Center for Regenerative Therapies TU Dresden

institute: TUD Dresden University of Technology

summary: The evolutionary expansion of the human neocortex reflects increased amplification of basal progenitors in the subventricular zone, producing more neurons during fetal corticogenesis. Here, we analyze the transcriptomes of distinct progenitor subpopulations isolated by a novel approach from developing mouse and human neocortex. We identify 56 genes preferentially expressed in human apical and basal radial glia that lack mouse orthologs. Among these, ARHGAP11B has the highest degree of radial glia-specific expression. ARHGAP11B arose from partial duplication of the Rho GTPase-activating-protein–encoding ARHGAP11A on the human lineage after separation from the chimpanzee lineage. Expression of ARHGAP11B in embryonic mouse neocortex promotes basal progenitor generation and self-renewal, and can increase cortical plate area and induce gyrification. Hence, ARHGAP11B may have contributed to evolutionary expansion of human neocortex.

overall_design: Gene expression profiles of mouse and human purified neocortical progenitor types and neurons were generated by RNA-seq and analyzed including inter- and intra-species comparison.

first: Marta

last: Florio

first: Wieland

last: Huttner


title: Differential susceptibility of human pleural and peritoneal mesothelial cells to asbestos exposure

city: Burlington

first: Julie

last: Dragon

state: VT

address: 95 Carrigan Dr

department: MMG

institute: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE

summary: We hypothesize that the observed differences in incidences of pleural and peritoneal malignant mesothelioma (MM) are the result of differences in the direct response of these cell types to asbestos rather than to differences mediated by the in vivo microenvironment. To test this hypothesis, we characterized cellular responses to asbestos in a controlled environment using high-throughput RNA sequence and other assays.

overall_design: Examination of asbestos-treated versus untreated mesothelial cells from four cell lines representing two tissue types in culture.

first: Arti

last: Shukla


title: The Sarcoglycan complex is expressed in the cerebrovascular system and is specifically regulated by astroglial Cx30 channels

city: Paris

first: Laurent

last: Jourdren

institute: École Normale Supérieure

summary: Astrocytes, the most prominent glial cell type in the brain, send specialized processes called endfeet around blood vessels and express a large molecular repertoire regulating the cerebrovascular system physiology. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction protein Connexin 43 and 30 (Cx43 and Cx30) allowing in particular for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. In this study, we addressed the specific role of Cx30 at the gliovascular interface. Using an inactivation mouse model for Cx30 (Cx30Δ/Δ), we showed that absence of Cx30 does not affect blood-brain barrier (BBB) organization and permeability. However, it results in the cerebrovascular fraction, in a strong upregulation of Sgcg encoding g-Sarcoglycan (SG), a member of the Dystrophin-associated protein complex (DAPC) connecting cytoskeleton and the extracellular matrix. The same molecular event occurs in Cx30T5M/T5M mutated mice, where Cx30 channels are closed, demonstrating that Sgcg regulation relied on Cx30 channel functions. We further characterized the cerebrovascular Sarcoglycan complex (SGC) and showed the presence of α-, β-, δ-, γ-, ε- and ζ- SG, as well as Sarcospan. Altogether, our results suggest that the Sarcoglycan complex is present in the cerebrovascular system, and that expression of one of its members, g-Sarcoglycan, depends on Cx30 channels. As described in skeletal muscles, the SGC may contribute to membrane stabilization and signal transduction in the cerebrovascular system, which may therefore be regulated by Cx30 channel-mediated functions.

overall_design: Comparison of 3-month-old Cx30 deleted mice against WT genetic background.

first: Anne-Cécile

last: Boulay

first: Bruno

last: Saubaméa

first: Salvatore

last: Cisternino

first: Corine

last: Blugeon

first: Martine

last: Cohen-Salmon


title: IGF2BP2/IMP2 Deficient Mice Resist Obesity through enhanced translation of Ucp1 mRNA and other mRNAs encoding Mitochondrial Proteins

city: Cambridge

first: Diego

last: Borges-Rivera

state: MA

address: 7 Cambridge Center

institute: Broad Institute

summary: Imp2 -/- mice were subjected to a number of characterization assays, including RNA-Seq of various tissues,

overall_design: Characterization of IMP2 -/- mice  including various metabolic measurements.

first: Ning

last: Dai

first: Joseph

last: Avruch


title: Perturbation of parent-specific gene expression during interspecific hybridization

city: Davis

first: Luca

last: Comai

state: Calif

address: 451 Health Sciences Dr

institute: UC Davis

summary: Interspecific hybridization often induces epigenetic remodeling that leads to transposon activation, gene expression changes, and loss of imprinting. These genomic changes can be deleterious and lead to postzygotic hybrid incompatibility. In Arabidopsis, loss of genomic imprinting of PHERES1 and presumed failure of Polycomb Repressive Complex is partially responsible for seed inviability observed in A. thaliana X A. arenosa interspecific hybrids. We used this species pair to further analyze the relationship between parent-specific gene expression and postzygotic hybrid incompatibility using two A. thaliana ecotypes, Col-0 and C24, with differential seed survival. We found that maternal imprinting was perturbed for PHERES1, HDG3, and six other genes in both A. thaliana hybrids and paternal imprinting was lost for MEDEA as observed previously. Three classes of retroelements; Sadhu, Athila, and Copia, maintained proper repression patterns suggesting some regulatory mechanisms are not disrupted early in development. We propose that early genome remodeling and loss of imprinting of seed development genes induces lethality in both compatible and incompatible hybrids.

sample_organism: Arabidopsis thaliana x Arabidopsis arenosa

overall_design: We examined gene expression in A. thaliana intraspecific hybrid crosses to determine normal patterns of maternal and paternal expression early in seed development.

first: Diana

last: Burkart-Waco

first: Kathie

last: Ngo

first: Meric

last: Lieberman


title: Target mRNAs induce tailing and trimming of Ago2-loaded miRNAs in neurons

first: Dimos

last: Gaidatzis

type: Non-coding RNA profiling by high throughput sequencing

summary: We show that target mRNAs trigger the non-templated addition of nucleotides (mainly adenosines and uridines) and shortening of their cognate miRNAs in primary hippocampal neurons. The induced effect is observable both in total RNA and in Ago2-associated RNA, demonstrating that the process is initiated in Ago2 bound miRNAs.

overall_design:
Illumina Small RNA sequencing was performed on rat hippocampal neurons infected with different target mRNAs in different conditions. The data includes 3 independent experiments:
1. Hippocampal neurons (DIV15) 6 days after transducing them with target mRNAs (driven by the Synapsin promoter) against different miRNAs. Samples are titled "Syn-Target". Samples transduced with targets containing extensive complementarity are labeled "Ext" and those with Seed complementarity are labeled "Seed".
2. Hippocampal neurons (DIV16) 7 days after transducing them with an inducible target mRNA against miR-132, and inducing for different times with Doxycycline. Samples are titled "Time-course"
3. Hippocampal neurons (DIV16) 7 days after co-transducing them with FLAG/HA-Ago2 plus an inducible target mRNA against miR-132, and inducing them for different times with Doxycycline. Samples are titled "Time-course_Ago2"

first: Manuel

last: de la Mata

first: Helge

last: Grosshans


title: Role of Fft3 in nuclear organization

city: Hamburg

first: Babett

last: Steglich

address: Martinistrasse 52

institute: Universitätsklinikum Hamburg-Eppendorf

type: Methylation profiling by genome tiling array

type: Genome binding/occupancy profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Microprocessor mediates transcription termination in long noncoding microRNA genes

city: Oxford

first: Nicholas

last: Proudfoot

address: South Parks Road

country: United Kingdom

institute: Sir William Dunn School of Pathology,  University of Oxford

summary: MicroRNA (miRNA) play a major role in the post-transcriptional regulation of gene expression. In mammals most miRNA derive from the introns of protein coding genes where they exist as hairpin structures in the primary gene transcript, synthesized by RNA polymerase II (Pol II). These are cleaved co-transcriptionally by the Microprocessor complex, comprising DGCR8 and the RNase III endonuclease Drosha, to release the precursor (pre-)miRNA hairpin, so generating both miRNA and spliced messenger RNA1-4. However, a substantial minority of miRNA originate from Pol II-synthesized long non coding (lnc) RNA where transcript processing is largely uncharacterized5. Here, we show that most lnc-pri-miRNA do not use the canonical cleavage and polyadenylation (CPA) transcription termination pathway6, but instead use Microprocessor cleavage both to release pre-miRNA and terminate transcription. We present a detailed characterization of one such lnc-pri-miRNA that generates the highly expressed liver-specific miR-1227. Genome-wide analysis then reveals that Microprocessor-mediated transcription termination is commonly used by lnc-pri-miRNA but not by protein coding miRNA genes. This identifies a fundamental difference between lncRNA and pre-mRNA processing. Remarkably, inactivation of the Microprocessor can lead to extensive transcriptional readthrough of lnc-pri-miRNA, resulting in inhibition of downstream genes by transcriptional interference. Consequently we define a novel RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

overall_design:
Chromatin associated RNA-seq from sicntrl,siDrosha,siDGCR8 treated Hela cells. Same for sicntrl and siDGCR8 from Huh7 cells. Nuclear polyA + and polyA- RNA-seq from sicntrl and siDGCR8 in HeLa cells.
Chromatin associated RNA-seq from siDicer treated Hela cells.

first: Ashish

last: Dhir

first: Somdutta

first: Catherine

last: Jopling


title: CHO smallRNA sequencing for microRNA annotation

city: Vienna

first: Andreas

last: Diendorfer

address: Muthgasse 18

department: Department of Biotechnology

country: Austria

institute: University of Natural Resources and Life Sciences

summary: MicroRNAs are small non-coding RNAs that play a critical role in post-transcriptional control of gene expression. Recent publications of genomic sequencing data from CHO cells provide new tools for the discovery of novel miRNAs in this important production system. The current release of the miRNA registry miRBase contains 307 mature miRNAs and 200 precursor sequences. We searched for evolutionary conserved miRNAs in recently published genomic data, derived from hamster and CHO cells, to further extend the list of known miRNAs. With our approach we could identify several dozens novel miRNAs and verify their expression with multiple next-generation sequencing experiments. In addition, a great amount of unexpressed miRNAs could be found, awaiting further confirmation by testing for their transcription in different Chinese hamster tissues.

overall_design: Two batch processes with 6 samples each

first: Fabian

last: Stiefel

first: Friedemann

last: Hesse


title: Role of Fft3 in nuclear organization [MNase-seq]

summary: In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3Δ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements at the borders of subtelomeres and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and components of the nuclear envelope by maintaining chromatin structure required for anchoring DNA insulators to nuclear pores.

overall_design: For MNase samples, duplicate mutant mononucleosome fractions were compared with duplicate WT mononucleosomes.

first: Annelie

last: Strålfors

first: Olga

last: Khorosjutina

first: Jenna

last: Persson

first: Agata

last: Smialowska

first: Jean

last: Javerzat

first: Karl

last: Ekwall


title: Genome-wide analysis of histone modifications in human endometrial stromal cells

first: Isao

last: Tamura


title: Effect of PRDM11 depletion in U2932 cells

city: Copenhagen

first: Anders

middle: H

last: Lund

address: Ole Maaloes Vej 5

department: Biotech Research and Innovation Centre

country: Denmark

institute: University of Copenhagen

summary: The PR-domain family e(PRDMs) encodes transcriptional regulators, several of which are deregulated in cancer. We found that loss of Prdm11 accelerates MYC-driven lymphomagenesis in the Eµ-Myc mouse model. Moreover, we show that patients with PRDM11-deficient diffuse large B cell lymphomas (DLBCLs) have poorer overall survival and belong to the non-Germinal Center B cell (GCB)-like subtype. Mechanistically, genome-wide mapping of PRDM11 binding sites coupled with transcriptome sequencing in human DLBCL cells evidenced that PRDM11 associates with transcriptional start sites of target genes and regulates important oncogenes such as FOS and JUN.  Hence, we characterize PRDM11 as a novel tumor suppressor controlling the expression of key oncogenes and add new mechanistic insight into B-cell lymphomagenesis.

overall_design: RNA-seq performed after knockdown of Prdm11

first: Cathrine

middle: K

last: Fog


title: Identification of chilling stress-responsive tomato microRNAs and their target genes by high-throughput sequencing and degradome analysis

city: Nanjing

first: xue

last: cao

address: Xuanwu District No. 1

institute: Nanjing Agricultural University

summary: To identify tomato miRNAs involved in chilling response, two small RNA libraries and two degradome libraries from chilling-treated(4°C/4°C for 1h，4h, 8h, 12h, 24h and 48h) and non-chilling-treated (25°C/20°C for 1h，4h, 8h, 12h, 24h and 48h)leaves of LA1777’ (S. habrochaites) seedlings were constructed. A total of 4342604 and 7231609 clean reads were obtained by high-throughput sequencing of the two libraries, respectively. 161 conserved miRNAs and 236 novel miRNAs were identified in the two librayies. Of these miRNAs, 192 were upregulated, whereas 205 were downregulated in response to chilling stress. Among these, 23 miRNAs and 26 miRNAs were significantly upregulated and downregulated in response to chilling stress, respectively.Through degradome sequencing,62 target genes were cleaved by 42 conserved miRNAs, while nine target genes were cleaved by nine novel miRNAs. Target gene functional analysis revealed that most target genes played positive role in the chilling response, primarily by regulating the expression of anti-stress proteins, antioxidant enzymes and genes involved in cell wall formation.

overall_design: tomato miRNA-seq and Degradome-seq after chilling

first: Xue

last: Cao

first: Zhen

last: Wu


title: Small RNA loading licenses Argonaute for assembly into a transcriptional silencing complex

status: Public on Mar 01 2015

first: Daniel

last: Holoch

state: Cedex 05

address: 26 rue d'Ulm

department: Genetics and Developmental Biology Unit

institute: Institut Curie, Centre de Recherche

summary: We analyzed the small RNAs bound to the fission yeast RITS and ARC complexes, and to Ago1 in wild-type and ARC mutant cells, to gain insight into the molecular role of ARC. We found that ARC contains longer heterochromatic siRNAs than RITS, and that ARC subunit Arb1 is required for loading small RNAs onto Ago1.

overall_design: Sequencing of 18-28 nt small RNAs from Tas3, Arb1 and Ago1 purifications.

first: D

last: Moazed


title: Cytosolic Hsp60 orchestrates the survival and inflammatory responses of vascular smooth muscle cells in injured aortic vessels by regulating NF-κB-dependent gene expression

city: Goyaongsi

first: Charny

last: Park

state: Gyeonggi-do

address: 323 Ilsan-ro,

country: South Korea

institute: National Cancer Center

summary: Pro-inflammatory response of VSMCs is triggered by endothelial damage and a causative step for thrombosis and neointimal thickening in the arterial vessels. Therefore, we investigate a role of cytosolic Hsp60 as a novel pro-inflammatory mediator in VSMCs.  Hsp60 was detected in the cytosol of VSMCs. The selective depletion of cytosolic Hsp60 in VSMCs reduced the IKK activation, repressed the induction of NF-κB-dependent pro-survival genes (MnSOD and Bfl-1/A1), and enhanced apoptotic death in response to TNF-α. Moreover, a quantitative RNA sequencing revealed that the expression of 75 genes among the 774 TNF-α-inducible genes was significantly reduced by the depletion of cytosolic Hsp60. In particular, the expression of pro-inflammatory cytokines/chemokines, such as CCL2, CCL20, and IL-6, was regulated by the cytosolic Hsp60 in VSMCs. Finally, the depletion of cytosolic Hsp60 markedly inhibited the neointimal thickening in the balloon-injured arterial vessels by inducing apoptotic cell death and inhibiting chemokine production.  This study provides the first evidence that cytosolic Hsp60 could be a therapeutic target for preventing inflammation-driven VSMC hyperplasia in the injured vessels.

overall_design: Hsp60 normal vs knockout with TNF-alpha treatment

first: Sang-Won

last: Kang

first: Sanghyuk

last: Lee

first: Jaesang

last: Kim

first: Mina

last: Choi

first: Boae

first: Doo-Jae

first: Dong-Hoon

first: Sunjoo

last: Jung


title: Phenotype specific analyses of p53 reveal distinct regulatory mechanism for chronically activated p53

first: Chandra

last: Chilamakuri

address: Robinson Way

institute: Cancer Research UK Cambridge Institute

summary: p53 ChIP seq and Histone mark ChIP Seq data in IMR90 (normal human diploid fibroblasts)

overall_design: Phenotypes were created by overexpression of RasG12V, E1A/RasG12V in IMR90 cells, growing IMR90 and 24 hour Etoposide treated IMR90 cells were subjected to p53 and histone mark ChIP Seq

first: Kristina

last: Kirschner

first: Shamith

last: Samarajiwa

first: Suraj

last: Menon


title: Targeting the MLL complex in castration-resistant prostate cancer [ChIP-seq]

city: Ann Arbor

first: Marcin

middle: Piotr

last: Cieslik

address: 500 S State St

department: Pathology

institute: University of Michigan

summary: Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. The development and progression to CRPC following androgen ablation therapy is predominantly driven by unregulated androgen receptor (AR) signaling. Despite the success of recently approved therapies targeting AR signaling, such as abiraterone and second-generation anti-androgens MDV3100 (enzalutamide), durable responses are limited, presumably due to acquired resistance. Recently, JQ1 and I-BET, two selective small molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit antiproliferative effects in a range of malignancies. Here we show that AR signaling-competent CRPC cell lines are preferentially sensitive to BET bromodomain inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ1. Like the direct AR antagonist, MDV3100, JQ1 disrupted AR recruitment to target gene loci. In contrast to MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR mediated gene transcription including induction of TMPRSS2-ERG and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer.

overall_design: Examination of ASH2L genome-wide binding in prostate cancer cells after AR stimulation.

first: Matthew

last: Iyer

first: Rohit

last: Malik


title: PRDM11 ChIP in U2932 cells

first: Anders

last: Lund

overall_design: 5 samples: PRDM11 Ab1, PRDM11 Ab2, RNA Polymerase II, IgG and input

first: Cathrine

last: Fog


title: Brg1 Modulates Enhancer Activation and Polycomb-mediated Repression in Mesoderm Differentiation [ChIP-exo]

city: San Francisco

first: Jeffrey

middle: Michael

last: Alexander

address: 1650 Owens St

institute: Gladstone Institutes

summary: We investigated the genome-wide occupancy changes in normal and Brg1-deleted mesoderm differentiation of mouse embryonic stem cells of chromatin regulators and histone modifications.

overall_design: ChIP exo for BRG1 in 2 conditions: Day4 THF (Control) and Day4 4OHT (Brg1-deleted).  1-2 replicates per condition.

first: Swetansu

last: Hota


title: The splicing factor PRP31 is involved in transcriptional gene silencing and stress response in Arabidopsis

city: Beijing

first: huanwei

last: huang

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

institute: NIBS

summary: To study how PRP31, ZOP1 and STA1 affect gene expression and pre-mRNA splicing, we performed RNA deep sequencing for the seedlings materials C24, prp31, zop1, and sta1 mutants.

overall_design: Compare the mRNA profiles of 10-day old seedlings materials of mutants (prp31, zop1 and sta1) to wild type C24 by Illumina suquencing.

first: Xinjian

last: He

first: Jinlu

last: Du

first: Huanwei

last: Huang


title: RNA-seq analysis of neonatal mouse cochlear hair cells

city: Houston

first: Andrew

last: Groves

address: 1 Baylor Plaza

department: Molecular and Human Genetics

institute: Baylor College of Medicine

summary: This study examined transcripts that are enriched in neonatal mouse cochlear hair cells. Hair cells were purified by FACS sorting for GFP fluorescence from the cochleas of transgenic mice in which the endogenous Atoh1 gene was fused with GFP

overall_design: Two replicates of GFP+ hair cells were compared with all other cochlear cell types that were GFP-


title: Next Generation Sequencing Facilitates Quantitative Analysis of  Bone Marrow Macrophages and Spleenic Macrophages Transcriptomes in mouse T cell acute lymphoblastic leukemia

city: Tianjin

first: Guo Guang

last: Zheng

state: Tianjin

address: Blood Hospital,288 Nanjing Road,Heping District

department: Institute of Hematology & Blood Hospital

institute: Chinese Academy of Medical Science& Peking Union  Medical College

summary: The goals of this study aim to reveal functional and phenotypic diversity of leukemia-associated macrophages in response to the microenvironmental cues in mouse T cell acute lymphoblastic leukemia

overall_design: Compare Transcriptomes of macrophages in T cell acute leukemia which are suggested as leukemia-associated macrophages (LAMs) with homeostasis

first: Xiao

last: Yang

first: ShaYan

last: Chen

first: Guoguang


title: PRDM11: a novel tumor suppressor


title: Genome wide miR-191 target profile determined by RIP and gene expression profiling

city: Austin

first: Vishy

address: 2500 Speedway Dr. MBB 3.212

department: Molecular Biosciences

institute: University of Texas at Austin


title: ChIP-seq mapping of histone modifications in CD14++ CD16- monocytes from septic disease patients

city: Giessen

first: Marek

last: Bartkuhn

state: Hessen

address: Aulweg 132

department: Biomedical Informatics and Systems Medicine

institute: Justus-Liebig-University Giessen

summary: Chromatin immunoprecipitation with antibodies specific for histone modifications H3K4me3, H3K9ac and H3K27me3 and subsequent high-throughput sequencing were performed on fixed chromatin from two septic disease patients.

overall_design: ChIP-seq

first: Sebastian

last: Weiterer


title: miR-191 regulates human cell proliferation and directly targets multiple oncogenes [seq]

summary: We constructed a genome wide target profile of hsa-miR-191 by sequencing RNA isolated from Ago2 immunoprecipitations and total RNA samples following miR-191 transfection

overall_design: Examination of mRNA levels in HeLa cells and Ago2 immunoprecipitations from HeLa cells following miR-191 mature duplex or control siRNA transfection

first: Damon

last: Polioudakis


title: SRSF2 is essential for hematopoiesis and its myelodysplastic syndromes-related mutations dysregulate alternative pre-mRNA splicing

city: La Jolla

first: Xiang-Dong

last: Fu

address: 9500 Gilman Drive, Room 231

department: CMM

institute: UC San Diego

summary: We report the biological function of Srsf2 in hematopoiesis in conditional knockout mouse models. Ablation of Srsf2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased hematopoietic stem/progenitor cells. Induced ablation of Srsf2 in adult Mx1Cre/ Srsf2flox/flox mice upon polyinosinic:polycytidylic acid injection demonstrated a significant decrease in lineage-/Sca+/cKit+ cells in bone marrow. To reveal the functional impact of MDS-associated mutations in SRSF2, we profiled global splicing responses on an MDS-L cell line using RASL-seq, and found that the P95H missense mutation and P95 to R102 in-frame 8 amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact Srsf2 is essential for the functional integrity of the hematopoietic system, and its mutations are likely key driver events to MDS.

overall_design: MDS-L cells (in triplicate) were transfected by srsf2 shRNA only, or pTRIPZ vectors containing both srsf2 shRNA and srsf2 mutants cDNA including P95H and P95 8 amino acid deletion as well as wild-type construct, followed by Dox induction. Total RNAs were extracted and been analyzed by RASL-seq.

first: Yukiko

last: Komeno

first: Jinsong

last: Qiu

first: Yu

last: Zhou

first: Hai-Ri

last: Li

first: Dong-Er

last: Zhang


title: SRD: a Staphylococcus Regulatory RNA Database

city: RENNES

first: Yoann

last: AUGAGNEUR

address: 2 Avenue du Pr Leon Bernard

department: Pharmaceutical Biochemistry

institute: University Rennes 1

summary: We report the use of Next Generation RNA Sequencing for confirming or improving initial identification of small regulatory RNA in Staphylococcus aureus to prodive a list of the most probable RNA molecules transcribed as independent units.

overall_design: Extraction of RNAs in exponential phase of growth in Newman and N315 strains

first: Mohamed

last: Sassi

last: Augagneur

first: Tony

last: Mauro

first: Lorraine

last: Ivain

first: Svetlana

last: Chabelskaya

first: Marc

last: Hallier

first: Olivier

last: Salou

first: Brice

last: Felden


title: Quantitative modeling of transcription factor binding specificities using DNA shape

city: New York

first: Namiko

last: Abe

department: Biochemistry and Molecular Biophysics

institute: Columbia University

summary: The SELEX-seq platform was used to generate DNA-binding affinity predictions for the human Max transcription factor.  This experiment was performed as part of a cross-validation study comparing the accuracy of DNA shape-augmented TF binding specificity models across two different platforms (SELEX-seq and gcPBM)

overall_design: Two rounds of SELEX were performed on Max protein as described in Slattery et al, Cell, 2011 (PMID 22153072).  Briefly, His-tagged Max was incubated with a randomized 16mer oligonucleotide library (GTTCAGAGTTCTACAGTCCGACGATCTGG[ACGT]{16}CCAGAACTCGTATGCCGTCTTCTGCTTG).  Max bound DNA was amplified and sequenced as described (Slattery et al, 2011).

first: Richard

last: Mann

first: Harmen

last: Bussemaker

first: Raluca

last: Gordân


title: RNA-Seq analysis to profile the transcriptome of Bone Marrow  Dendritic Cells exposed to different antigen delivery systems

city: Naples

first: Piergiuseppe

last: De Berardinis

state: Italy

address: Via P. Castellino, 111

department: Institute of Protein Biochemistry

country: Italy

institute: National Research Council

summary: Here we report the analysis of the transcriptome of bone marrow-derived dendritic cells exposed to the different antigen delivery systems to dissect the molecular mechanisms that orchestrate the immune response upon  vaccination. RNA-Sequencing revealed that exposure of dendritic cells to diffent antigen display vehicules causes the deregulation of molecules involved in the immune response, at a different extent, depending on the carrier. In vaccinology, patterns of gene expression induced by priming antigen presenting cells (APCs), could be used to predict immunogenicity and the type of T-helper cell polarization, and/or to helpin selecting an appropriate adjuvant to administer in the vaccine formulation.

overall_design: Bone marrow precursor at day 7 of GM-CSF culture were exposed or not to LPS deprived bacteriophage, bacteriophage modified to be targeted to DC or to the E2 protein scaffold

first: P

first: L

last: D'Apice

first: M

last: Trovato

first: R

last: Sartorius

first: A

last: Ciccodicola

first: V

last: Costa


title: Single mammalian cells compensate for differences in cellular volume and DNA copy number through independent global transcriptional mechanisms

city: Philadelphia

first: Arjun

last: Raj

state: PA

address: 210 S. 33rd St., Ste. 240 Skirkanich Hall

department: Bioengineering

institute: University of Pennsylvania

summary: We performed single-cell and bulk transcriptome profiling in two different human cell lines. We performed single-cell RNA sequencing in live and fixed cells.

overall_design: Single cell RNA sequencing of live and fixed cells, bulk RNA sequencing in two cell lines.

first: Olivia

last: Padovan-Merhar

first: Gautham

middle: P

last: Nair

first: Andrew

last: Biaesch

first: Andreas

last: Mayer

first: Steven

last: Scarfone

first: Shawn

middle: W

last: Foley

first: Angela

middle: R

last: Wu

last: Churchman

first: Abhyudai

last: Singh


title: Embryonic small RNA and RNA profiles of Tribolium castaneum and Drosophila oocytes

city: Pasadena

first: Maria

last: Ninova

address: 1200 E California Blvd

department: Division of Biology and Biological Engineering

institute: Calfironia Institute of Technology

summary: Next generation sequencing analysis of the small RNA and RNA profiles of successive time intervals during T. castaneum embryogenesis, and Drosophila unfertilized oocytes

sample_organism:
Tribolium castaneum
Drosophila melanogaster
Drosophila virilis

overall_design: Total RNA was extracted from oocytes precisely staged embryos at 25°C and small RNA or stranded polyA-RNA libraries were generated according to the manufacturer's instructions

first: Sam

last: Griffiths-Jones

first: Matthew

last: Ronshaugen


title: Phenotypic responses of differentiated asthmatic human airway epithelial cultures to rhinovirus

first: Yongsheng

last: Huang

state: MI

address: 100 Washtenaw Ave

department: Bioinformatics

summary: We report the application of RNA sequencing technology for high-throughput profiling of gene expression responses to human rhinovirus infection at 24 hours in air-liquid interface human airway epithelial cell cultures derived from 6 asthmatic and 6 non-asthmatic donors.  RNA-seq analysis identified sets of genes associated with asthma specific viral responses.  These genes are related to inflammatory pathways, epithelial remodeling and cilium assembly and function, including those described previously (e.g. CCL5, CXCL10 and CX3CL1), and novel ones that were identified for the first time in this study (e.g. CCRL1, CDHR3).   We concluded that air liquid interface cultured human airway epithelial cells challenged with live HRV are a useful in vitro model for the study of rhinovirus induced asthma exacerbation, given that our findings are consistent with clinical data sets.  Furthermore, our data suggest that abnormal airway epithelial structure and inflammatory signaling are important contributors to viral induced asthma exacerbation.

overall_design: Differentiated air-liquid interface cultured human airway epithelial cell mRNA profiles from 6 asthmatic and 6 non-asthmatic donors after 24 hour treatment with either HRV or vehicle control were generated by deep sequencing, using Illumina HiSeq 2000.

first: Jianwu

last: Bai


title: Simultaneous transcriptome analysis of Colletotrichum gloeosporioides and tomato fruits response reveals novel fungal-fruit arm and defense strategies

city: Rehovot

first: Gilgi

last: Friedlander

address: Hertzel st

institute: Weizmann Institute of Science

summary: The simultaneous gene fungal-fruit expression at appressoria, quiescence and necrotrophic stages in C. gloeosporioides-tomato fruit interaction were characterized by Illumina NGS. Fungal appressoria stage on green fruit showed stage-specific transcription and was accompanied by massive fruit transcriptional defense response. The quiescent fungal transcriptome showed activation of chromatin remodeling genes while the fruit response continued a highly integrated and massive up-regulation of defense genes. The necrotrophic stage showed a dramatic shift in up-regulation of C. gloeosporioides pathogenicity factors and a susceptive fruit response that shows activation of the salicylic acid pathway culminating in cell death and anthracnose disease. Simultaneous fungal-fruit transcriptome analysis deepens our perception of the unfolding fungal-fruit arms and defenses race.

sample_organism:
Solanum lycopersicum
Colletotrichum gloeosporioides

overall_design: Tomato fruits cv. Mottel were inoculated with 7 µl, 106 conidia/ml at 30 inoculation spots per fruit on 5 different fruits per biological replicate with seven biological replicates per treatment. RNA for the appressoria and quiescent stages were collected 19 and 100 hpi of mature green tomato fruits inoculation, respectively. For necrotrophic stage growth a 10 mm2 of fruit cuticle was removed and inoculated with 70 µl of a conidial suspension (106 conidia/ml). Three inoculation spots on each of 10 different fruits were inoculated per biological replicate with seven biological replicate per treatment. RNA for necrotrophic stage evaluation was collected 48 hpi by removing 0.5 mm depth of tissue. Control fruits were similarly treated and processed by mock inoculation.

first: Alkan

last: Noam

first: Friedlander

last: Gilgi

first: Ment

last: Dana

first: Prusky

last: Dov

first: Fluhr

last: Robert


title: Aberrant chromatin acetylation in MLL-AF9 leukemia mediates the response to HDAC inhibition (ChIP-Seq)

city: Madrid

first: Sara

last: Alvarez

state: Madrid

address: C/Faraday 7

country: Spain

institute: NIMGenetics

summary: We studied the chromatin modification patterns induced by the presence of the MLL-AF9 fusion protein in a model of human hematopoietic stem/progenitor cells (HSPC) transduced with retrovirus expressing MLL-AF9cDNA (HSPC-MA9). Comparative ChIP-seq analysis between HSPC-MA9 and control HSPC, revealed a massive hyperacetylation of histones that was consistent with the transcriptional profile in the presence of MLL-AF9 fusion protein. Furthermore, we identified 66 MLL-AF9 targets, and found that H4ac was present along with H3K4me3 and H3K79me2 chromatin marks in over 50% of the MLL-AF9 target genes.

overall_design: Examination of histone aceylation and methylation changes upon expression of MLL-AF9 fusion protein in human hematopoietic stem/progenitor cells.

first: Alba

last: Maiques-Diaz

first: Alvaro

last: Eguileor

first: Mahesh

last: Shrestha

first: Gonzalo

last: Gómez López

first: Mark

last: Wunderlich

first: Ana

last: del Rio-Machin

first: Javier

last: Santoyo-Lopez

first: Xabier

last: Agirre

first: James

middle: C

last: Mulloy

first: Juan

last: Cigudosa


title: Aberrant chromatin acetylation in MLL-AF9 leukemia mediates the response to HDAC inhibition

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

overall_design: Refer to individual Series


title: Brg1 Modulates Enhancer Activation and Polycomb-mediated Repression in Mesoderm Differentiation

first: Jeffrey

last: Alexander

state: CA


title: Drosophila melanogaster S2 cells nucleosome occupancy data after simulation by heat killed Staphylococcus aureus

status: Public on Feb 28 2015

city: Tallahassee

first: Jonathan

last: Dennis

state: FL

address: 319 Stadium Dr

department: Biological Science

institute: Florida State Unversity

summary: The innate immune response is among the strongest genomic responses and is conserved across all metazoa.  Although transcription during the innate immune response has been well studied, the associated chromatin reorganizations are largely uncharacterized.  Here we show that Drosophila S2 cells stimulated with Staphylococcus aureus display a dynamic change in genome-wide nucleosome occupancy and sensitivity. We found a widespread and transient nucleosomal loss peaking at 30 minutes post stimulation, and we demonstrated that the regulatory potentials of nucleosomes differ following stimulation. In addition, we identified differentially sensitive nucleosomes with response-specific potentials. Our results provide high-resolution nucleosome-distribution maps of the fly genome, revealing chromatin's role in: the innate immune response to Gram-positive bacteria, response-specific regulatory-factor binding, and nucleosome sensitivity. We identify functional chromatin regulatory features associated with immune response, and lay a foundation for a framework linking general and locus-specific roles for nucleosomes in immune regulation.

overall_design: Drsophila S2 cells at 0hr, 30minutes, 1hr and 4hr post heat-killed Staphylococcus aureus stimulation

first: Yingxue

last: Ren

first: Daniel

middle: L

last: Vera

first: Kimberly

last: Hughes


title: Brg1 Modulates Enhancer Activation and Polycomb-mediated Repression in Mesoderm Differentiation [RNA-Seq]

summary: We investigated the global gene expression changes in normal and Brg1-deleted mesoderm differentiation of mouse embryonic stem cells.

overall_design: RNAseq analysis of poly-adenylated RNA levels for 3 conditions: Day2 of differentiation, Day4 THF (Control), and Day4 4OHT (Brg1-deleted).  2 replicates per condition.


title: Deregulated microRNAs in triple-negative breast cancer revealed by deep sequencing

city: Taipei

first: King-Jen

last: Chang

address: No.1, Changde St., Zhongzheng Dist.

department: Department of Surgery

country: Taiwan

institute: National Taiwan University Hospital

summary: Twenty-four triple-negative breast cancer and 14 adjacent normal tissues were collected from breast cancer patients during surgeries at National Taiwan University Hospital (NTUH, Taipei, Taiwan). All triple-negative breast cancer samples were invasive ductal carcinomas (IDC) and were negative in immunohistochemical statuses of ER, PR, and HER2 receptors, as confirmed by professional pathologists. Treatment procedure of all patients followed the National Comprehensive Cancer Network (NCCN) guideline. All samples were neoadjuvant-free and were collected before systemic chemotherapy treatments. Written informed consent was obtained from all patients who participated in this study. Using human tissues for research in this study was approved by the institutional review board at NTUH. A novel set of 25-miRNA signature identified in this study was able to effectively distinguish between triple-negative breast cancer and adjacent normal tissues. Moreover, we documented the first evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNA genes in triple-negative breast cancer.

overall_design: In the present study, a panel of 24 triple-negative breast cancer and 14 adjacent normal tissue samples were examined for the presence of deregulated miRNA genes using the high-throughput sequencing technology. Total RNA was extracted from the triple-negative breast cancer and adjacent normal samples for preparation of small RNA libraries. Each small RNA library was constructed from total RNA of each sample using the SOLiD Total RNA-Seq Kit (Applied Biosystems, Foster City, CA, USA). Upon completion of polymerase chain reaction (PCR) amplification, small RNA libraries were purified using the SOLiD Library Micro Column Purification Kit (Applied Biosystems) and hybridized to the template beads using the SOLiD EZ bead system (Applied Biosystems). The template beads were amplified and deposited onto subtract for ligation sequencing by SOLiD 4 System (Applied Biosystems).

first: Wen-Hung

last: Kuo


title: Haploinsufficiency, but not defective paternal 5mC oxidation, accounts for the developmental defects of maternal Tet3 knockouts

city: Boston

first: Li

last: Shen

address: 200 Longwood Ave

institute: HHMI/Boston Children's Hospital/Harvard Medical School

summary: DNA demethylation of paternal genome in zygotes takes place in various mammals including mice and human. Recent studies have revealed that this is achieved through Tet3-mediated iterative oxidation of 5-methylcytosine (5mC) coupled with replication-dependent dilution. Tet3-mediated paternal DNA demethylation is believed to be required for mouse development given that Tet3 heterozygous embryos, derived by fertilizing Tet3 knockout (KO) oocytes with wild-type (WT) sperms, exhibit 5mC oxidation defects and embryonic sublethality, Here we demonstrate that the sublethality phenotype of the maternal KO mice is caused by haploinsufficiency of Tet3, but not by defective paternal 5mC oxidation. We found that Tet3 heterozygous mice derived from crosses of heterozygous father or mother with WT mice also exhibit sublethality phenotype similarly to Tet3 maternal KO mice. Importantly, embryos reconstituted with WT paternal nuclei that bypassed 5mC oxidation develop to term and grow to adulthood normally. Genome-scale DNA methylation analysis of the maternal KO zygotes and blastocysts demonstrated that hypermethylation caused by the depletion of maternal Tet3 is largely diminished by the blastocyst stage. Our study thus not only demonstrates that Tet3-mediated paternal 5mC oxidation is dispensable for mouse development but also suggests the existence of a compensation mechanism in preimplantation embryos that can compensate for the defective 5mC oxidation in zygotes.

overall_design: This data set includes RRBS data of wild-type and maternal Tet3 KO zygotes and blastocysts (C57BL/6J x CAST/EiJ)

first: Azusa

last: Inoue

first: Shogo

last: Matob

first: Yi

last: Zhang


title: Brg1 Modulates Enhancer Activation and Polycomb-mediated Repression in Mesoderm Differentiation [ChIP-Seq]

city: San Francisco

address: 1650 Owens St

institute: Gladstone Institutes

summary: We investigated the genome-wide occupancy changes in normal and Brg1-deleted mesoderm differentiation of mouse embryonic stem cells of chromatin regulators and histone modifications.

overall_design: ChIP analysis for BRG1-FLAG, SUZ12, H3K27ac, or H3K27me3 in 2 conditions: Day4 THF (Control) and Day4 4OHT (Brg1-deleted).  2-3 replicates per condition.


title: De Novo Assembly and Characterization of Stress Transcriptome in a salinity-Tolerant Variety CS52 of Brassica juncea

city: New Delhi

first: Ashwani

last: Pareek

state: Delhi

address: New Mehrauli Road

department: School of Life Sciences

country: India

institute: Jawaharlal Nehru University

summary: Oilseed mustard, Brassica juncea, exhibits high levels of genetic variability for salinity tolerance. To obtain the global view of transcriptome and investigate the molecular basis of salinity tolerance in a salt-tolerant variety CS52 of B. juncea, we performed transcriptome sequencing of control and salt-stressed seedlings. De novo assembly of 184 million high-quality paired-end reads yielded 42,327 unique transcripts longer than 300 bp with RPKM ≥1. When compared with non-redundant proteins, we could annotate 67% unigenes obtained in our study. Based on the mapping to expressed sequence tags (ESTs), 52.6% unigenes are novel compared to EST data available for B. juncea and constituent genomes. Differential expression analysis revealed altered expression of 1469 unigenes in response to salinity stress. Of these, 587, mainly associated with ROS detoxification, sulfur assimilation and calcium signaling pathways, are up regulated. Notable of these is RSA1 (SHORT ROOT IN SALT MEDIUM 1) INTERACTING TRANSCRIPTION FACTOR 1 (RITF1) homolog up regulated by >100 folds in response to stress. RITF1, encoding a bHLH transcription factor, is a positive regulator of SOS1 and several key genes involved in scavenging of salt stress-induced reactive oxygen species (ROS). Further, we performed comparative expression profiling of key genes implicated in ion homeostasis and sequestration (SOS1, SOS2, SOS3, ENH1, NHX1), calcium sensing pathway (RITF1) and ROS detoxification in contrasting cultivars, B. juncea and B. nigra, for salinity tolerance. The results revealed higher transcript accumulation of most of these genes in B. juncea var. CS52 compared to salt-sensitive cultivar even under normal growth conditions. Together, these findings reveal key pathways and signaling components that contribute to salinity tolerance in B. juncea var. CS52.

overall_design: We report transcriptome sequencing of two-weeks-old seedlings of B. juncea var. CS52 under normal growth conditions (CTRL) and in response to salinity stress (SS) using Illumina paired-end sequencing

first: Rita

last: Sharma

first: Manjari

last: Mishra

first: Brijesh

last: Gupta

first: Chirag

last: Parsania

first: Sneh

last: Singla-Pareek


title: Sensitive detection of chromatin-altering polymorphisms reveals autoimmune disease mechanisms

city: Singapore

first: Ricardo

middle: Cruz-Herrera

last: del Rosario

address: 60 Bipolis St, #02-01 Genome

department: Computational and Systems Biology

country: Singapore

institute: Genome Institute of Singapore

summary: Most disease associations detected by genome-wide association studies (GWAS) lie outside coding genes, but very few have been mapped to causal regulatory variants. Here, we present a method for detecting regulatory quantitative trait loci (QTLs) that does not require genotyping or whole-genome sequencing. The method combines deep, long-read chromatin immunoprecipitation–sequencing (ChIP-seq) with a statistical test that simultaneously scores peak height correlation and allelic imbalance: the genotype-independent signal correlation and imbalance (G-SCI) test. We performed histone acetylation ChIP-seq on 57 human lymphoblastoid cell lines and used the resulting reads to call 500,066 single-nucleotide polymorphisms de novo within regulatory elements. The G-SCI test annotated 8,764 of these as histone acetylation QTLs (haQTLs)—an order of magnitude larger than the set of candidates detected by expression QTL analysis. Lymphoblastoid haQTLs were highly predictive of autoimmune disease mechanisms. Thus, our method facilitates large-scale regulatory variant detection in any moderately sized cohort for which functional profiling data can be generated, thereby simplifying identification of causal variants within GWAS loci.

overall_design: We applied our method to 57 cell lines from a single population group. We used the resulting sequence data for variant calling, and validated calls using an independent platform. We then identified histone acetylation QTLs (haQTLs) using a novel statistical test that requires no prior genotype information and combines peak height and allelic imbalance data across the 57 individuals. Transcription factor binding site analysis was used to independently support the functionality of haQTLs. Finally, we examined the association between haQTLs and SNPs associated with human phenotypes.

first: Jeremie

last: Poschmann

first: Sigrid

last: Rouam

first: Eileen

last: Peng

first: Chiea

last: Khor

first: Martin

last: Hibberd

first: Shyam

last: Prabhakar


title: Gene and MicroRNA Expression Responses to Exercise; Relationship with Insulin Sensitivity

status: Public on Feb 27 2015

city: Scottsdale

first: Lawrence

middle: J

last: Mandarino

state: Arizona

address: 13400 E. Shea Boulevard

department: Researcg

country: USA

institute: Mayo Clinic in Arizona

summary: Healthy individuals on the lower end of the insulin sensitivity spectrum also have a reduced gene expression response to exercise for specific genes. The goal of this study was to determine the relationship between insulin sensitivity and exercise-induced gene expression in an unbiased, global manner.

overall_design: Euglycemic clamps were used to measure insulin sensitivity and muscle biopsies were done at rest and 30 minutes after a single acute exercise bout in 14 healthy participants. Changes in mRNA expression were assessed using microarrays [GSE43219], and miRNA analysis was performed in a subset of 6 of the participants using sequencing techniques.

first: Dawn

last: Coletta


title: RNA-seq in squamous cell carcinoma stem cells in the presence or absence of TGF-beta signaling

first: Naoki

last: Oshimori

state: New York

address: 1230 York Avenue

institute: The Rockefeller University/HHMI

summary: We performed RNA-seq on purified squamous cell carcinoma stem cells (SCC-SCs) from primary mouse skin tumors transduced with TGF-beta reporter.

overall_design: SCC-SCs were purified based on cell surface marker expression integrin alpha6 and CD44, after lineage negative selection, and separated by fluorescent TGF-beta reporter expression.

first: Elaine

last: Fuchs


title: A Circult Mechanism for Differentiating Positive and Negative Associations

first: Suzuko

last: Yorozu

state: Massachusettes

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: The ability to differentiate stimuli predicting positive or negative outcomes is critical for survival, and perturbations of emotional processing underlie many psychiatric disease states.  Different neuronal populations of the basolateral amygdala complex (BLA) encode fearful or rewarding associations, but the molecular identity of these functionally distinct populations of BLA neurons remained unknown. Here, we show that BLA neurons projecting to the nucleus accumbens (NAc-projectors) or the centromedial amygdala (CeM-projectors) underwent opposing synaptic changes following fear or reward conditioning. The photostimulation of NAc projectors supported positive reinforcement while photostimulation of CeM projectors mediated negative reinforcement. In search of defining molecular characteristics of these functionally-distinct BLA neuronal populations, we compared gene expression profiles of NAc- and CeM-projectors.

overall_design: For comparison of gene expression profiles of NAc- and CeM-projectors, we conducted two independent RNA sequencing experiments. In experiment-1, a total of n=9 samples (n=4 NAc- and n=5 CeM-projectors) are analyzed. In experiment-2, a total of n=8 samples (n=4 NAc- and n=4 CeM-projectors) are analyzed.

first: Jesse

middle: M

last: Gray


title: MiRNA23B REGULATES SELF-RENEWAL AND CHEMORESISTANCE PROPERTIES OF COLON CANCER STEM CELLS

city: Newark

first: Bruce

last: Boman

state: DE

address: 4701, ogletown-stanton road

department: CTCR

institute: Helen F Graham Cancer Center and Research Institute

summary: MiRNAs have been identfied to play an important role in cancer stem cells. MiR23b is differentially expressed in various forms of cancer including colorectal cancer as compared to their normal counterparts. MiR23b regulates various aspects of cell behaviour such as differentiation, apoptosis and motility. The goal of the study was to identify the novel role of miR23b in self-renewal property of colon cancer stem cells via regulation of its candidate target mRNAs. To address this aim, HT29 colon cancer cells were transfected with miR23b Precursor, Antimir and their respective controls. RNA SEQ analysis of the cells with altered levels of miR23b assisted in the identification of interesting mRNA targets influenced by miR23b expression and involved self-renewal pathways.

overall_design: HT29 cells with altered levels of miR23b was subjected to RNA SEQ analysis to identify differential expresssion of mRNA targets of mIR23b

first: Vignesh

last: Viswanathan

first: Monica

last: Accerbi

first: Skye

last: Schmidt


title: Sexual cell fate reprogramming in the ovary by DMRT1

city: Minneapolis

first: Micah

last: Gearhart

state: MN

address: 6-160 Jackson Hall, 321 Church St.

department: Genetics, Cell Biology and Development

institute: University of Minnesota

summary: Transcription factors related to the insect sex determination gene Doublesex (DMRT proteins) control sex determination and/or sexual differentiation in diverse metazoans. They also are implicated in transitions between sex-determining mechanisms during vertebrate evolution. In mice Dmrt1 is required for male gonadal differentiation in somatic cells and germ cells. DMRT1 also maintains male gonadal sex: its loss, even in adults, can trigger sexual fate reprogramming in which male Sertoli cells transdifferentiate into their female equivalents - granulosa cells - and testicular tissue reorganizes to a more ovarian morphology. Here we use a conditional Dmrt1 transgene to show that Dmrt1 is not only necessary but also sufficient to specify male cell identity in the mouse gonad. DMRT1 expression in the ovary silenced the female sex-maintenance gene Foxl2 and reprogrammed juvenile and adult granulosa cells into Sertoli-like cells, triggering formation of structures resembling male seminiferous tubules. DMRT1 can silence Foxl2 even in the absence of the testis-determining genes Sox8 and Sox9. mRNA profiling found that DMRT1 activates many testicular genes and downregulates ovarian genes and single cell RNA-seq in transdifferentiating cells identified dynamically expressed candidate mediators of this process. Strongly upregulated genes were highly enriched on chromosome X, consistent with sexually antagonistic functions. This study provides an in vivo example of single gene reprogramming of cell sexual identity. Our findings suggest a reconsideration of mechanisms involved in human disorders of sexual development (DSD) and empirically support evolutionary models where loss or gain of Dmrt1 function promotes establishment of new vertebrate sex determination systems.

overall_design: RNA-Seq (3 conditions, 2 replicates per condition) and Single Cell RNA-Seq (68 individual cells and 1 bulk cell sample)

first: David

last: Zarkower


title: Gene Networks Specific for Innate Immunity Define Post-traumatic Stress Disorder

first: Michael

middle: S

last: Breen

state: NY

address: 1468 Madison Ave

department: Depts. of Psychiatry, Genetics and Genomic Sciences

institute: Ichan School of Medicine at Mount Sinai


title: Neuronal CRTC-1 governs systemic mitochondrial metabolism and lifespan via a catecholamine signal

city: Ann Arbor

first: Ana

middle: Rodrigues

last: Grant

address: 100 Washtenaw Avenue

department: Computational Medicine & Bioinformatics

institute: University of Michigan

summary: Low energy states delay aging in multiple species, yet mechanisms coordinating energetics and longevity across tissues remain poorly defined. The conserved energy sensor AMP-activated protein kinase (AMPK) and its corresponding phosphatase calcineurin modulate longevity via the ‘CREB regulated transcriptional coactivator (CRTC)-1 in C. elegans. We show that CRTC-1 specifically uncouples AMPK/calcineurin mediated effects on lifespan from pleiotropic side effects by reprogramming mitochondrial and metabolic function. Strikingly, this pro-longevity metabolic state is regulated cell-nonautonomously by CRTC-1 in the nervous system. CRTC-1/CREB act antagonistically with the functional PPARα ortholog, NHR-49 to promote distinct peripheral metabolic programs. Neuronal CRTC-1 drives mitochondrial fragmentation in distal tissues and suppresses the effect of AMPK on systemic mitochondrial metabolism and longevity via a cell-nonautonomous catecholamine signal. These results demonstrate that transcriptional control of neuronal signals can override enzymatic regulation of metabolism in peripheral tissues. Central perception of energetic state therefore represents a target to promote healthy aging.

overall_design: Experiment was performed with three biological replicates. Gravid adults grown at 20¡C on 100 mm NG plates seeded with OP50-1 E. coli were collected and treated with hypochlorite to release eggs. Eggs were incubated overnight in M9 media to obtain L1 synchronized populations. One thousand L1 larvae were grown on a 100 mm NG plate seeded with OP50-1 E. coli. Worms were harvested for RNA extraction when L4 larval stage was reached. Animals were collected and washed extensively with M9 media to remove bacteria. Worms were then snap frozen in liquid nitrogen. RNA was extracted by five freeze/thaw cycles in Qiazol then purified by RNeasy mini kit (Qiagen). RNA quality was checked using an Agilent Technologies 2100 Bioanalyzer. All samples had an RNA integrity number of 10. cDNA libraries were prepared from 4 ugs of total RNA using the TruSeq RNA Sample Preparation v2 kit (Illumina). 50-cycle paired-end sequencing was performed on an Illumina HiSeq 2000 by the Harvard Biopolymer Core. Read quality was evaluated with FASTQC. Adapter sequences and poor quality bases (<20) were trimmed and filtered with CUTADAPT, resulting in a median of 44 million reads per replicate. These were aligned to the C. elegans genome (ce6, WS238) using TopHat version 2.0.8 (Kim et al., 2013), with a median 35 million reads mapped in proper pairs. The number of reads mapping to each gene was counted with htseq-count. Genes with less than 1 Count Per Million Reads (CPM) were discarded from further analysis. Counts were normalized for sequencing depth and RNA composition across all samples with edgeR (Robinson et al., 2010). Genes were tested for differential expression between each mutant strain and wild-type using edgeR’s glm method. For each comparison, genes with less than 5 CPM were filtered and those with at least 50% change and False Discovery Rate (FDR) of 1% or less were considered differentially expressed.

first: Kristopher

last: Burkewitz

first: Ianessa

last: Morantte

first: Heather

last: Weir

first: Robin

last: Yeo

first: Yue

last: Zhang

first: Frank

last: Huynh

first: Olga

last: Ilkayeva

first: Matthew

middle: D

last: Hirschey

first: William

last: Mair


title: CAGE-Seq of gonads and carcasses from D. melanogaster and D. pseudoobscura

status: Public on Feb 26 2015

city: Bethesda

first: Brian

last: Oliver

state: MD

address: 50 South Drive

department: LBG

institute: NIDDK, NIH

summary:
We performed CAGE-Seq on dissected ovaries and testes as well as female and male carcasses of two species of Drosophila (D. melanogaster and D. pseudoobscura).  These data are used to map transcription start sites.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

sample_organism:
Drosophila melanogaster
Drosophila pseudoobscura

overall_design: For both species, D. melanogaster and D. pseudoobscura, CAGE-Seq was performed on dissected gonads (with reproductive tracts) and remaining carcasses from wild-type adults, using Illumina GAIIx (SRA Study SRP001602, Bioproject PRJNA36315).  There are eight samples.  Only Drosophila melanogaster testis samples were performed in biological duplicate.  See Chen ZX et al. Genome Res 2014, 24(7):1209-23.

first: Zhen-Xia

last: Chen