title: HER2-positive breast cancer cells resistant to trastuzumab and lapatinib lose reliance upon HER2 and are sensitive to the multitargeted kinase inhibitor sorafenib

status: Public on Dec 15 2010

city: Biella

first: caterina

last: Peraldo Neia

state: Biella

address: via Malta 3

department: Laboratory of  Cancer Genomics

institute: Fondazione Edo ed Elvo Tempia

summary:
HER2 targeting with trastuzumab has changed the prognosis of breast cancer patients carrying amplification and/or overexpression of this oncogene. Despite this progress, however, resistance to trastuzumab occurs in the vast majority of patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show antitumor activity in a limited proportion of patients, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, due to the high proportion of patients that fail to respond to these alternative strategies, it is reasonable to assume that cells escaping HER2 targeting may rely on alternative pathways not involving HER2 to sustain their growth. Their knowledge is relevant for exploiting new therapies. To investigate this hypothesis we generated a human HER2 overexpressing breast cancer cell line resistant to trastuzumab and lapatinib (T100) and we have characterized it genetically and molecularly.  In T100 cells, the previously proposed mechanisms of resistance to HER2 inhibitors did not explain cell escape. Notably, silencing HER2 by shRNA did not affect the growth of T100 cells, suggesting loss of reliance upon HER2. Among the alternative pathways that we explored, altered levels of the antiapoptoptic proteins Mcl-1 and Survivin were observed. This suggested the possibility of targeting resistant cells with the multitarget inhibitor sorafenib. Moreover, sorafenib, nearly inactive in parental cells, strongly inhibited the in vitro growth of T100 cells.  In conclusion, we provide a new model where the activation of HER2 independent mechanisms sustains the growth of tumor cells, significantly increasing the sensitivity to sorafenib.

Keywords: HER2, breast cancer, resistance, trastuzumab, lapatinib, sorafenib

overall_design: Total RNA was isolated using TRIZOL reagent (Life Technologies, Gathersburg, MD) and quantified by Bioanalyzer 2100 (Agilent Technologies); following the isolation procedure, mRNA was amplified starting from 1μg using MessageAmp II aRNA Amplification kit (Ambion Inc. Austin TX, USA). Ammino-allyl modified nucleotides were incorporated in in vitro transcription step according to the manufacturer’s protocol.  Labeling was performed using NHS (N-hydroxysuccinimidyl) ester Cy3 or Cy5 dies (GE Healthcare Europe GMBH, Upsala - Sweden) able to react with the modified RNA. At least 5 μg of mRNA, for each sample, were labeled and purified with columns respectively. The Dye-Swap replication procedure was applied, in order to increase the accuracy of results. Samples were hybridized on 44K glass arrays (Agilent Technologies). Arrays were scanned and the images obtained were analyzed by the Feature Extraction software Agilent (version 9.5) and the text files were then processed using the Bioconductor package Limma (Linear models for microarray analysis).

first: Giorgio

last: Valabrega

first: Sonia

last: Capellero

first: Giuliana

last: Cavalloni

first: Gianluca

last: Zaccarello

first: Annalisa

last: Petrelli

first: Giorgia

last: Migliardi

first: Andrea

last: Milani

first: Caterina

last: Peraldo-Neia

first: Loretta

last: Gammaitoni

first: Francesco

last: Favero

first: Aldo

last: Moggio

first: Silvia

last: Giordano

first: Massimo

last: Aglietta

first: Filippo

last: Montemurro


title: Gene expression profiles of different subpopulations of hepatocellular carcinoma

status: Public on Dec 28 2010

city: Shanghai

first: Ying

last: Hou

address: Yueyang Road 320

country: China

institute: Institute of Neuroscience, China Academy of Science

summary: To demonstrate CD133+CD44+ and CD133+CD44- subpopulations of hepatocellular carcinoma as distinct subgroups, we have employed whole genome microarray expression profiling as a discovery platform to reveal the gene profiles of different subgroups and identify genes responsible for the enhanced metastatic potentials of CD133+CD44+ tumor cells. CD133+CD44+ and CD133+CD44- tumor cells were isolated from three human metastatic hepatocellular carcinoma specimens. A 76-gene consensus signature was identified that distinguished between CD133+CD44+ and CD133+CD44- subgroups. CD133+CD44+ and CD133+CD44- subgroups from different patients were well clustered as two distinct classes according to this signature, and many genes in this signature were reported involved in tumor metastasis. Expression of four genes (CCL4, DKK3, CCR5 and MMP12) from this signature was confirmed in another three metastatic HCC specimens by real-time PCR.

overall_design: CD133+CD44+ and CD133+CD44- subpopulations of hepatocellular carcinoma were isolated from three metastatic hepatocellular carcinoma specimens by flow cytometry. A total of 30K to 50K cells for each subgroup was obtained for each microarray.

first: Yizheng

last: Wang


title: Transcriptional response to hypoxia of normal and rheumatoid arthritis synovial fibroblasts

status: Public on Dec 10 2010

city: Madrid

first: Manuel

middle: J

last: Del Rey

address: Avda Córdoba s/n

department: Grupo de Enfermedades Inflamatorias y Autoinmunes

country: Spain

institute: Instituto de Investigación Hospital 12 de Octubre

summary: Inflammatory tissues are characterized by low oxigen concentrations (hypoxia). These conditions are very different from that usually present in tissue cultures where transcriptomic profiles of human fibroblasts from inflammatory tissues have been previously analysed. The aim of this study was to characterize the changes on gene expression induced by hypoxia in human synovial fibroblasts. We used microarray expression profiling in paired normoxic and hypoxic cultures of healthy and rheumatoid arthritis (RA) synovial fibroblasts (HSF and RASF). Hypoxia induces significant changes on the expression of large groups of genes in both HSF and RASF. The hypoxic and normoxic profiles are also different between both groups. These data demonstrate that hypoxia induces significant changes on gene expression in HSF and RASF and identify differences between RASF and HSF.

overall_design: Synovial fibroblasts obtained from 6 patients with rheumatoid arthritis (RASF) and 6 sex and age matched adult healthy donors (HSF) were used. SF cultures were incubated for 22 hours under normoxic or hypoxic (0.5% O2) conditions. Nine experiments per group were performed, single experiments with three SF lines, and duplicated in other three lines per group. All 18 normoxia-hypoxia experiments (36 microarray data) were used for paired analysis of the changes induced by hypoxia in HSF or RASF.

first: Elena

last: Izquierdo

first: Alicia

last: Usategui

last: Gonzalo

first: Francisco

last: Blanco

last: Acquadro

first: José

middle: L

last: Pablos


title: Worsening of Obesity and Metabolic Status Yields Similar Molecular Adaptations_Subcutaneous and Visceral Adipose Tissue

status: Public on Dec 07 2010

city: Toulouse

first: Nathalie

last: Viguerie

address: Avenue Poulhes

country: France

institute: INSERM

summary:
Context: It is not known whether biological differences reported between subcutaneous (SAT) and visceral (VAT) adipose tissue depots underlie the pathogenicity of visceral fat.
Objective: We compared SAT and VAT gene expression according to obesity, visceral fat accumulation, insulin resistance and presence of the metabolic syndrome.
Design: Subjects were assigned into 4 groups (lean, overweight, obese and obese with metabolic syndrome).
Setting: Subjects were recruited at a university hospital.
Patients: 32 women were included.
Main Outcome Measures: Anthropometric measurements, euglycemic hyperinsulinemic clamps, blood analyses and computed tomography scans were performed and paired samples of SAT and VAT were obtained for DNA microarray-based gene expression profiling.

overall_design:
Whole transcriptome analysis was performed using DNA microarrays on subcutaneous and visceral adipose tissue of the selected 8 subjects per group (lean, overweight, obese and obese with metabolic syndrome).
A total of 64 array hybridizations were performed. Cy5 dye was incorporated into adipose tissue RNA samples while a reference RNA pool made of a mix of commercial human liver, adipose tissue and skeletal muscle RNA was labelled with Cy3 dye (Applied Biosystems/Ambion).
Samples were hybridized to Agilent 44k whole human genome microarrays with over 41,000 60-mer oligonucleotides from unique human sequences and transcripts.


title: Propionibacterium acnes as a frequent inflammatory agent involved in prostate cancer

status: Public on Dec 22 2010

city: Berlin

first: Hans-Joachim

last: Mollenkopf

address: Charitéplatz 1

country: Germany

institute: Max-Planck-Institute for Infection Biology

summary: Prostate cancer is the second leading cause of male cancer deaths in the United States and Europe. Current evidence implicates an inflammatory mechanism as a cause of prostate carcinogenesis. Here we show that the bacterium Propionibacterium acnes (P. acnes) is prevalent in prostate glandular tissues: 70% of benign hyperplasia and 81% of cancer tissue samples tested positive for the bacterium. Live P. acnes bacteria were isolated from cancerous prostates and co-cultured with epithelial prostate cells to confirm they were cell invasive. Transcriptome and ELISA studies revealed that P. acnes induced a strong inflammatory response in prostate cells, resulting in the secretion of cytokines and chemokines such as interleukin (IL)-6 and IL-8. In addition, P. acnes triggered the COX prostaglandin and the plasminogen-matrix metalloproteinase (MMP) pathways. Strikingly, long-term exposure of non-tumorigenic prostate cells to P. acnes resulted in loss of E-cadherin, altered betacatenin levels and localization, increased cellular migration, and conferred anchorage- independent growth. Our work adds to the growing body of work highlighting the presence of viruses and bacteria in cancerous prostate tissues and strongly suggests that P. acnes infection could lead to malignant transformation of prostate cells.

overall_design: Microarray experiments were performed as dual-color hybridizations. To compensate for dye-specific effects, a dye-reversal color-swap was applied. Ratio profiles comprising color-swap hybridizations were combined in an error-weighted fashion to create ratio experiments. A 1.5–fold change expression cut-off for ratio experiments was applied together with anti-correlation of color-swap ratio profiles rendering the microarray analysis highly significant (P-value > 0.01), robust and reproducible.

first: Lina

last: Fassi-Fehri

first: Holger

last: Brueggemann

first: Tim

last: Mak

first: Thomas

middle: F

last: Meyer


title: Comparative analysis of mouse cardiac gene expression:  diet, sex, and disease

status: Public on Dec 01 2010

city: Boulder

first: Leslie

last: Leinwand

state: CO

address: 3415 Colorado Ave

department: MCD Biology and BioFrontiers Institute

country: USA

institute: University of Colorado at Boulder

summary:
The perception that soy food products and dietary supplements will have beneficial effects on heart health has led to a massive consumer market. However, we have previously noted that diet has a profound effect on disease progression in a genetic model of hypertrophic cardiomyopathy (HCM). In this model, a soy-based diet negatively impacts cardiac function in male mice.
Given the frequent correlation between functional changes and transcriptional changes, we investigated the effect of diet (soy- vs. milk-based) on cardiac gene expression and how it relates to the additional factors of sex and disease. We found that gene expression in the heart is altered more by diet than by sex or an inherited disease. We also found that the healthy male heart may be sensitized to dietary perturbations of gene expression in that it displays a gene expression profile more similar to diseased hearts than to healthy female hearts. These observations may in part account for divergence in HCM phenotypes between males and females and between diets.

overall_design: Hearts from male and female wild-type or HCM C57BL/6 mice fed either a soy or casein-based diet were excised at 2 months of age. Total RNA was extracted from left ventricles. Biotin-labeled amplified RNA was hybridized to MG_U74Av2 Affymetrix microarrays.

first: Elizabeth

middle: D

last: Luczak

first: Kristen

middle: K

last: Barthel

first: Brian

last: Stauffer

first: John

middle: P

last: Konhilas

first: Tom

middle: H

last: Cheung

middle: A


title: Transcriptional responses to nanoparticulate matter in human colon cancer cells.

city: Salt Lake City

first: Philip

last: Moos

state: UT

department: Pharmacology & Toxicology

institute: University of Utah

summary:
This study evaluated transcriptional effects of particles smaller than 100 nm of carbon black, SiO2, Al2O3, TiO2, ZnO and Fe203.  Since there is concern that inflammation may increase the uptake and/or toxicity of ultrafine particles, we evaluated the transcriptional responses without or with a TNFα pretreatment to mimic an inflammatory state.
The most pronounced transcriptional response resulted from ZnO treatement, another response was to TNFα pre-treatment. We identified stress responses, responses to Zn ions, but did not observe a consistent proinflammatory response as evaluated by Gene Ontology of the genes that altered expression most as identified by significance analysis.

Keywords: colon cancer cells - response to distinct nanoparticulate

overall_design: Two colon cancer cell types (RKO and CaCo-2) were pretreated with 100 ng/ml BSA or TNFα for 1 hr, the media was exchanged and with media containing the nanoparticulate, and RNA was collected after 4 hrs.  Generally, the RNA from 4 independent samples were combined for one sample on the microarray, the exceptions were the TNFα treated controls where 6 independent samples were combined in 2 sets of 3 for the microarray labeling and hybridizations.


title: Early target genes of aflatoxin B1 in human hepatic cells

city: Rennes

first: rozenn

last: jossé

address: 2 av Prof Léon Bernard

institute: INSERM U991

summary:
Comparison of gene expression profiles induced by the mycotoxin, aflatoxin B1 (AFB1), in primary human hepatocytes and HepaRG cells.
Initial mechanisms involved in the complex multistep process leading to malignant transformation by chemicals remain largely unknown. We have analysed changes in gene expression profiles in primary human hepatocytes and differentiated human hepatoma HepaRG cells after a 24 h treatment with 0.05 or 0.25µM aflatoxin B1 (AFB1), a potent genotoxic hepatocarcinogen.

overall_design: Three independent biological replicates of HepaRG cell cultures and two pools of three primary human hepatocyte cultures each, were investigated. Cells were treated with 0.05 or 0.25µM AFB1 for 24 h.

first: R

last: Jossé

first: A

last: Rogue

first: C

last: Lambert


title: Control of chromatin structure by Spt6 has different consequences in coding and regulatory regions

city: Montréal

first: François

last: Robert

state: Québec

address: 110, avenue des Pins Ouest

department: Chromatin and Genomic Expression

country: Canada

institute: Institut de recherches cliniques de Montréal (IRCM)

type: Genome binding/occupancy profiling by array

summary: Spt6 is a highly conserved factor that carries out important functions in transcription and chromatin structure. To gain new insights into Spt6, we measured nucleosome occupancy along Saccharomyces cerevisiae chromosome III in an spt6 mutant and found that the level of nucleosomes is greatly reduced accross some but not all coding regions. In addition, genome-wide location analyses of RNA polymerase II showed that the nucleosome loss in the spt6 mutant occurs over highly-transcribed genes. Unexpectedly, the effects of the spt6 mutation on nucleosome levels did not correlate with its effects on mRNA levels, suggesting that Spt6 plays distinct roles in controlling chromatin structure across coding regions and in transcriptional regulation. We studied one case of transcriptional regulation by Stp6, at the CHA1 gene, and showed that regulation likely occurs by Spt6 controlling the position of the +1 nucleosome. These results, along with previous studies, suggest that Spt6 regulates transcription by controlling chromatin structure over regulatory regions, and its effects on nucleosome levels over coding regions likely serve and independent function.

overall_design: In order to examine the genome-wide localization of RNAPII and Spt6 in Saccharomyces cerevisiae, RNAPII and Spt6 along with associated DNA sequences were immunoprecipitated using anti-8WG16 and anti-HA antibodies, respectively. The RNAPII and Spt6 chromatin immunoprecipitation was performed in duplicate from WT cells as described below. The extracted DNA was hybridized to a DNA microarray containing an average of 4 probes per kilobase across the whole yeast genome. The combined datasets are available in the supplemental files of the related publication.

first: Iva

last: Ivanovska

first: Pierre-Étienne

last: Jacques

first: Oliver

last: Rando

first: Francois

first: Fred

last: Winston


title: Effect of valproic acid  on hematopoiesis: A functional profiling and cell phenotyping approach

city: Seoul

first: Marc

last: Diederich

address: Gwanak-ro, Gwanak-gu, 1

department: College of Pharmacy

country: South Korea

institute: Seoul National University

summary: Analysis of the effects of valproic acid (VPA) on chronic myelogenous leukemia K562 cells. This study attempts to elucidate the effects of VPA on cell homeostasis and hematopoietic differentiation pathways in this cell line.

overall_design: We used ten experimental conditions comparing valproic acid-treated and untreated cells at time points 2, 6, 10, 48 and 72 hrs respectively. Experiments were performed in three biological replicates including a dye swap (represented by replicate 3, for each timepoint).

first: Sebastien

last: Chateauvieux

first: Serge

last: Eifes

first: Franck

last: Morceau

first: Michael

last: Schnekenburger

first: Christina

last: Grigorakaki

first: Estelle

last: Henry

first: Mario

last: Dicato


title: A critical role of microRNAs in human pulmonary arterial hypertension. miR-204: a novel therapeutic target

status: Public on Dec 16 2010

city: Lausanne

first: Eric

middle: R

last: Paquet

state: Vaud

address: Station 15  CH - 1015

department: Life sciences

country: Switzerland

institute: EPFL

type:
Expression profiling by array
Expression profiling by RT-PCR

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

overall_design: Refer to individual Series


title: Expression Profiling: during in vitro neural differentiation from mES cells.

city: Kobe

first: Daisuke

last: Kamiya

address: 2-2-3 Minatojima-Minamimachi, Chuo

country: Japan

institute: RIKEN CDB

summary: GeneChip-based screen for genes induced in the initial phase of neural differentiation from ES cells.

overall_design: We established an efficient system for the in vitro neural differentiation of mouse ES cells involving floating aggregate culture in growth-factor-minimized medium (serum-free culture of embryoid body-like aggregates; SFEB, http://www.ncbi.nlm.nih.gov/pubmed/15696161). We use this culture system, Sox1-GFP ES cells (http://www.ncbi.nlm.nih.gov/pubmed/12923295) were differentiated with SFEB culture method for 1-5 days. Then, differentiated cells were sorted with Sox1 (neuroectodermal marker) or E-cadherin (undifferentiation marker) by FACS. And analyzed these cell populations by using Affymetrix Murine Genome U74 Version 2 Arrays (U74Av2, U74Bv2 and U74Cv2).

first: Lars

middle: M

last: Jakt

first: Mitsuhiro

last: Okada


title: Neural stem and progenitor cells shorten S-phase upon commitment to neuron production

status: Public on Nov 30 2010

city: Dresden

first: Yoko

last: Arai

address: Pfotenhauerstrasse 108

department: MPI-CBG

institute: Max Planck Institute

summary: The dorsal telencephalon from E14.5 Tis21–GFP heterozygous mice embryos was subjected to microarray analysis. To identify genes whose expression were changed in Tis21–GFP positive S phase cells compared to Tis21–GFP negative S phase cells, Tis21–GFP positive or negative S phase cells labeled with a vital dye were separated by FACS corresponding to the GFP expression and DNA contents of the cells.

overall_design: 30 telencephalic hemispheres from 15 embryos of the Tis21–GFP heterozygous mice at E14.5 were dissected to collect approximately 200,000 S phase cells either from Tis21–GFP positive or negative for one replicate. Four independent experiments were performed from 3 different pregnant mice at least for each experiment set. Finally three best experiment sets were used for data analyses.

first: Britta

last: Schilling

first: Wieland

last: Huttner


title: DNA Methylation Profiling of Glioblastoma: Impact on Gene Expression and Clinical Outcome (Agilent Expression Study)

status: Public on Jan 19 2011

first: Amandine

last: Etcheverry

address: 2 rue Henri Le Guilloux

institute: CNRS UMR 6290 - IGDR

summary: Genome-scale measurements of DNA methylation levels are necessary to decipher the epigenetic events involved in glioblastoma aggressive phenotype, and to guide new therapeutic strategies. In that purpose, we performed a whole genome integrative analysis of the methylation and expression profiles for 40 newly diagnosed glioblastoma patients. We have also screened for associations between CpG sites methylation levels and overall survival in a cohort of 50 patients uniformly treated with radiotherapy and chemotherapy with concomitant and adjuvant temozolomide (STUPP protocol).  The methylation analysis identified 616 CpG sites differentially methylated between glioblastoma and control brain, a quarter being differentially expressed in a concordant way. Among these concordant CpG sites, 13 genes displayed, within our glioblastoma cohort, an inverse correlation between promoter methylation and expression levels: B3GNT5, FABP7, ZNF217, BST2, OAS1, SLC13A5, GSTM5, ME1, UBXD3, TSPYL5, FAAH, C7orf13, and C3orf14. The expression of these genes may be tightly regulated by epigenetic mechanisms.   The survival analysis identified six CpG sites associated with overall survival. The SOX10 promoter methylation status (two CpG sites) stratifies the patients in a way similar to MGMT with improved performance based on Area Under the Curve criteria (0.78 vs. 0.71, p-value < 5.10-4). The methylation status of FNDC3B, TBX3, DGKI, and FSD1 promoters identify patients with MGMT methylated tumors non-responding to STUPP treatment (p-value < 1.10-4). These markers have a potential impact on therapeutic decision.

overall_design: 40 glioblastoma samples and 6 control brain samples were analysed. 2 distinct series of hybridizations were carried out, each containing GBMs and control brains.

first: Jean

last: Mosser


title: Genopal: a novel platform for focused microarray analysis

status: Public on Nov 10 2010

city: Suita

last: Okuzaki

state: Osaka

address: Yamadaoka 3-1

department: Immunology Frontier Research Center

institute: Osaka univ.


title: Agilent expression analysis of LNCaP cells following 5'aza-treatment

status: Public on Nov 15 2010

city: Ann Arbor

first: Jung

last: Kim

state: MI

address: 1400 E. Medical Center Drive

institute: University of Michigan

summary:
Beginning with precursor lesions, aberrant DNA methylation marks the entire spectrum of prostate cancer progression.  We mapped the global DNA methylation patterns in selected prostate tissues and cell lines using Methylplex-Next Generation Sequencing (M-NGS).  Hidden Markov Model based next generation sequence analysis identified ~68,000 methylated regions per sample.  While global CpG Island (CGI) methylation was not differential between benign adjacent and cancer samples, overall promoter CGI methylation increased from ~12.6% in benign samples to 19.3% and 21.8% in localized and metastatic cancer tissues, respectively.  We found distinct patterns of promoter methylation around transcription start sites, where methylation occurred not only on the CGIs, but also on flanking regions and CGI sparse promoters.  Among the 6,691 methylated promoters in prostate tissues, 2481 differentially methylated regions (DMRs) are cancer specific, including numerous novel DMRs.   A novel cancer specific DMR in WFDC2 promoter showed heavy methylation in cancer (17/22 tissues, 6/6 cell lines), but not in the benign tissues (0/10) and normal PrEC cells.  Integration of LNCaP DNA methylation and H3K4me3 data suggested an epigenetic mechanism for alternate transcription start site utilization and these modifications segregated into distinct regions when present on the same promoter.  Finally, we observed differences in repeat element methylation, particularly LINE-1, between ERG gene fusion positive and negative cancers.  This comprehensive methylome map will further our understanding of epigenetic regulation in prostate cancer progression.

In this data, genes methylated in LNCaP and significantly overexpressed after 5'Azacytidine treatment of LNCaP cells are assessed by Agilent gene expression microarray as a validation for gene repression mechanism due to methylation change.

overall_design: [Gene expression] Two-condition experiment, control DMSO-treated vs. 5'aza-treated LNCaP at two time points (@24 and 48 hours) in replicates.

first: Sunita

last: Shankar

first: Saravana

last: Dhanasekaran

first: Arul

last: Chinnaiyan


title: DSIF and RNA polymerase II CTD Phosphorylation Coordinate the Recruitment of Rpd3S to Actively Transcribed Genes

status: Public on Nov 28 2010

first: François

last: Robert

type: Genome binding/occupancy profiling by genome tiling array

summary: Histone deacetylase Rpd3 is part of two distinct complexes: the large (Rpd3L) and small (Rpd3S) complexes. While Rpd3L targets specific promoters for gene repression, Rpd3S is recruited to ORFs to deacetylate histones in the wake of RNA polymerase II, to prevent cryptic initiation within genes. Methylation of histone H3 at lysine 36 by the Set2 methyltransferase is thought to mediate the recruitment of Rpd3S. Here, we confirm by ChIP-Chip that Rpd3S binds active ORFs. Surprisingly, however, Rpd3S is not recruited to all active genes, and its recruitment is Set2-independent. However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive. Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes. Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain.

first: Simon

last: Drouin

first: Louise

last: Laramée

first: Pierre-Étienne

last: Jacques

first: Audrey

last: Forest

first: Maxime

last: Bergeron

first: Francois


title: Human fetal penile smooth muscle cells (hfPSCM): Control vs PCB mixtures treatments

status: Public on Nov 09 2010

city: Rome

first: Sabrina

last: Tait

state: Italy

address: Viale Regina Elena 299

department: Dpt. Infectious Diseases

country: Italy

institute: Istituto Superiore di Sanità

summary: The effects exerted by three mixtures of Polychlorinated Biphenyls (PCB), one featuring dioxin-like (DL) and two featuring non dioxin-like (NDL) congeners, on human fetal corpora cavernosa cells representative of a major male endocrine-sensitive organ, have been evaluated by gene expression profiling. PCB congeners concentrations used were derived from human internal exposure data to explore the impact of the adult body burden on a fetal tissue. Cell exposure were performed for 72h mimicking a chronic exposure.

overall_design: Experiment conditions: control, three treatments with PCB-Mix1, PCB-Mix2 or PCB-Mix3. Biological replicates: 3 control replicates, 3 replicates per treatment. Technical replica: 2 (dye swap) per biological replica. Direct comparison design (control vs treated samples) in 12/16 arrays (biological replicates 1 and 2); Loop design in 4/16 arrays (biological replicate 3).


title: Molecular Signature of response and potential pathways related to resistance to the HSP90 inhibitor, 17-AAG, in breast cancer.

status: Public on Oct 12 2010

first: Beatriz

last: Martinez

address: Calle Melchor Fernandez Almagro 3

department: Human Cancer Genetics

institute: Spanish National Cancer Research Centre (CNIO)

summary: We have done transcriptional profiling of 17-AAG treated (24h, 48H) breast cancer cell lines to understand  the mechanisms of the drug action and indicate novel set of pharmacodynamic biomarkers of the drug response as well as of resistance

overall_design: Two-condition experiment, Untreated vs. Treated sensitive cells. Phenotype replicates: 8 untreated replicates, 14 treated replicates.

first: Magdalena

last: Zajac

first: Gonzalo

last: Gomez-Lopez

first: Javier

last: Benitez


title: Comparative transcription profiling of peripheral blood mononuclear cells from vasculitis patients

first: Daisuke

summary: Peripheral blood mononuclear cells (PBMCs) from 13 vasculitis patients expressed varying gene profiles.

overall_design: A total of 13 sets of two color arrays were performed and each individual array included a target sample and a control sample.

first: Hiroshi

last: Nojima

first: Tomonori

last: Ishii

first: Shigeto

last: Kobayashi

last: Hashimoto


title: Prostate Cancer Field Effect

status: Public on Oct 18 2010

city: Seattle

first: Ilsa

last: Coleman

state: WA

address: 1100 Fairview Ave N, E2-112

department: Human Biology

institute: Fred Hutchinson Cancer Center

summary: In this study, comparison of gene expression profiles in benign epithelia from men with prostate cancer to those of men without prostate cancer reveal differences in several genes associated with prostate cancer.

overall_design: Custom Agilent 44K whole human genome expression oligonucleotide microarrays were used to profile benign epithelium from prostate needle biopsies from 15 men with high grade(Gleason 8-10) prostate cancer and 14 age- and BMI-matched controls. All samples were laser-capture microdissected and total RNA isolated and amplified prior to hybridization against a common reference pool of prostate tumor cell lines

first: Michael

middle: C

last: Risk

first: Beatrice

middle: S

last: Knudsen

first: Ruth

last: Dumpit

first: Alan

last: Kristal

first: Nolwenn

last: LeMeur

first: Robert

last: Gentleman

first: Lawrence

last: True

first: Peter

last: Nelson

first: Daniel

middle: W

last: Lin


title: Expression and regulation of corticotropin-releasing factor receptor type 2β in developing and mature mouse skeletal muscle.

status: Public on Nov 02 2010

city: Houston

first: Scott

middle: Andrew

last: Ochsner

state: TX

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary: The purpose of the study was to further understand the molecular mechanisms mediating the effect of Crhr2 signaling on insulin sensitivity in skeletal muscle. To that end we compared the gene expression profile of skeletal muscle obtain from both Crhr2 KO and WT littermates using gene expression microarray.

overall_design: RNA was extracted from skeletal muscle of 4 Crhr2 KO and 4 WT littermates. RNA from 2 KO or 2 WT mice was pooled and analyzed using Affymetrix U74Av2 GeneChips.

first: Wylie

last: Vale

first: Alon

last: Chen


title: The forced loss of Epstein Barr virus from Burkitt's lymphoma cells: uncomplemented cells vs BART miRNA complemented cells.

status: Public on Oct 01 2010

city: Madison

first: Bill

last: Sugden

state: WI

address: 1400 University Avenue

department: Oncology

institute: University of Wisconsin-Madison

summary: Transcriptional profiling of Burkitt lymphoma cells engineered to inducibly express dominant negative EBNA1 (dnEBNA1), which forces the loss of Epstein Barr virus (EBV). Profiles are made of cells in which all of EBV is lost (dnEBNA1 on) or all of EBV except the BART miRNAs is lost (dnEBNA1 on, BART miRNAs ectopically expressed).

overall_design: This is a 2-condition experiment: uncomplemented cells (EBV evicted with dnEBNA1), and BART-complemented cells (EBV evicted with dnEBNA1, BART miRNAs expressed ectopically). There are 3 biological replicates for each condition.

first: David

middle: T

last: Vereide


title: Defining Cell Fate And Embryonic Genome Activation By Global Single-Cell cDNA Analysis of Blastomeres From 5-to 8-Cell Human Embryos.

status: Public on Oct 30 2010

city: Valencia

first: Amparo

last: Galan

address: Avda Autopista del Saler 16, 3

institute: Prince Felipe Research Centre (CIPF)

summary: To determine blastomere fate and embryonic genome activation (EGA) at 5- to 8-cell stage human embryos by global gene expression profile of amplified cDNA from blastomeres at the single cell level

overall_design: Forty-nine blastomeres from 5-, 6- and 8-cell human embryos were analyzed through whole genome wide analysis following an efficient cDNA amplification protocol (Kurimoto et al., 2007) with slight modifications. Single biopsied blastomeres were also compared with two amplified inner cell masses and two trophectoderms from blastocysts.

last: Galán

last: Montaner

first: Maria

middle: E

last: Póo

first: Joaquín

last: Dopazo

first: Carlos

last: Simón


title: ChIP-chip from HCT116 cells treated with MMS and LNCaP cells with ATF3

city: Chiyodaku

first: Yujiro

last: Tanaka

state: Tokyo

address: 2-3-10 Kandasurugadai

department: School of Biomedical Science

institute: Tokyo Medical and Dental University

summary:
Maintenance of genetic integrity is essential for survival of all organisms. Activating transcription factor 3 (ATF3) is a member of the c-AMP response element binding (CREB)/ATF  family of transcription factors, and is highly inducible by various stress conditions including DNA damage. However, downstream targets and molecular basis underlying pleiotropic effects of ATF3 on the cell fate have been largely unknown. To identify ATF3 targets in the human genome, we carried out chromatin immunoprecipitation-microarray (ChiP-on-chip) and knockdown-expression profiling analysis using two models where ATF3 was either transiently induced or constitutively expressed. We show that ATF3 binds to an unexpectedly large number of targets; 5,984 promoters in HCT116 cells treated with an alkylating agene methyl methanesulfonate (MMS) and 1,423 promoters in LNCaP cells constitutively expressing ATF3. Importantly, targets of MMS-induced ATF3 are highly enriched not only for CREB/ATF motifs but also for binding sites of several stress sensors including DDIT3/CHOP, Egr1, and c-Ets which are concomitantly induced by MMS. Stress-induced ATF3 affects broad but select biological processes including cell cycle, cell death, adhesion, biosynthesis, and receptor signaling pathways. In addition, ATF3 binds to as many as 40% of the p53 targets and preferentially enhances MMS-induced activation of proapoptotic genes such as DR4, DR5, and PUMA, consistent with the proapoptotic effect of ATF3. These data shed new light on the co-regulatory function of ATF3 in the stress-induced transcription factor network. The comprehensive list of genomic targets of ATF3 will facilitate further understanding the role of ATF3 in determining life and death of cells under both physiological and tumour-associated stress conditions.
Maintenance of genetic integrity is fundamental to survival of all organisms. DNA damage can be caused by various agents in environment and elicits complex responses in the cell. ATF3 is one of the transcription factors activated by various stress conditions including DNA damage, and has been shown to have pleiotropic effects on life and death of cells depending on the context of experimental conditions. It has been largely unknown, however, which genes and pathways are regulated by stress-induced ATF3. Here we attempted to answer this question by chromatin immunoprecipitation-microarray analysis of  downstream targets of ATF3. We show that ATF3 binds to an unexpectedly large number of promoters (nearly 6,000) in a human colorectal cancer cell lineHCT116 treated with an alkylating agent methyl methanesulfonate. Interestingly, the ATF3 targets are highly enriched for binding sites of other stress sensors shedding light on a transcriptional co-regulatory network of DNA damage response. We further show that ATF3 regulates expression of genes in select biological processes including cell cycle, cell death, adhesion, metabolism, signal transduction, and the p53 pathway. The comprehensive list of ATF3 targets provides new insight into a  highly inter-connected network of stress-induced transcription factors around ATF3.

overall_design:
ChIP-chip samples: Comparison of ATF3-IP and whole genome DNA (control)
Gene expression samples: HCT116 cells pre-transfected with either control siRNA or ATF3 knockdown siRNA and stimulated by methyl methanesulfonate (MMS) for 0, 6, 12, and 24 hours

first: Aya

last: Nakamura

first: Masaki

last: Morioka

first: Shoko

last: Inoue

first: Mimi

last: Adachi

first: Kazuhiko

last: Yamada

first: Kenji

last: Taketani

first: Junya

last: Kawauchi

first: Hiroshi

first: Shigetaka

last: Kitajima


title: Subcutaneous adipose tissue (SAT) gene expression patterns between weight control and regaining weight subjects

city: Toulouse

first: Nathalie

last: Viguerie

address: Avenue Poulhes

institute: INSERM

summary: Objective: This work aimed at identifying and characterizing differences in subcutaneous adipose tissue (SAT) gene expression patterns between subjects succeeding weight control against subjects regaining weight 6 months after a low calorie diet program. Methods: Weight-reduced obese subjects from 8 European countries were randomized into 4 diets differing in protein and glycemic index. In addition to the anthropometric and plasma parameters, SAT biopsies were taken at the beginning (CID2) and the end (CID3) of the weight maintenance intervention. Pan-genomic cDNA microarrays were used to define SAT gene expression profiles at both CID2 and CID3 of 22 subjects succeeding weight maintenance (successful subjects; mean weight change= -2.6 ± 1.2 kg) and 22 subjects regaining weight (unsuccessful subjects; mean weight change= 3.9 ± 1.3 kg) among the 4 dietary arms. Results: Differences in SAT gene expression patterns between successful and unsuccessful groups were mainly due to weight variations rather to differences in diet macronutrient composition. An ANCOVA analysis with total energy intake at CID3 as covariant led us to the definition of 1339 differential genes. Functional analysis of differential genes showed that cellular growth and proliferation, inflammation, cell death, cancer, cellular function and maintenance were the main biological processes involving these differential genes.

overall_design: Overall design: SAT transcriptome was defined at the beginning (CID2) and at the end (CID3) of the dietary intervention program using a common reference design: Cyanine-5 dye was incorporated into all SAT samples, while a reference RNA pool made of the mix of commercial human liver, adipose tissue, and skeletal muscle RNA was labeled with cyanine-3 dye. Samples were hybridized to Agilent 44K whole human genome microarrays. Evolution in gene expression patterns after dietary intervention was tested in terms of the ratio after/before dietary intervention (CID3/CID2) ratio.

first: Adriana

last: Marquez-Quiñones

last: Mutch

first: Ping

last: Wang

first: Edwin

last: Mariman

first: Cyrille

last: Debard

first: Hubert

last: Vidal

first: Marion

last: Combes

first: Balbine

last: Roussel

first: Dominique

last: Langin

first: Consortium

last: Diogenes


title: Expression profiles of human osteoblasts directly and indirectly damaged by chemotherapy

status: Public on Sep 30 2010

city: Huntington

first: James

last: Denvir

state: WV

address: One John Marshall Drive

department: Biochemistry and Microbiology

institute: Marshall University School of Medicine


title: Expression data from PPARg WT and KO macrophages

status: Public on Sep 24 2010

city: Madrid

first: Mercedes

last: Ricote

address: Darwin 3

institute: CNB-CSIC

summary: PPARg is a nuclear receptor that plays an important role in lipid metabolism, homeostasis and immunity. Microarray analysis of gene expression was performed in macrophages from WT and PPARg KO mice. Differentially expressed genes were selected for further analysis.

overall_design: RNA from WT and PPARg KO macrophages was purified for hybridization on Affymetrix microarrays. Peritoneal macrophages were harvest from WT and PPARg KO mice 3 days after intraperitoneal injection of 2.5ml of 3% thioglycollate.

first: John

last: Welch

first: Christopher

last: Glass


title: Human osteoblast treated with Melphalen

summary: Global expression profile of human osteoblast treated with melphalen, compared to human osteoblast cells treated with diluent control

overall_design: Universal reference design with four biological replicates for the treated cells and four biological replicates for the untreated cells. Stratagene Universal Reference RNA was used as the common reference for all arrays.

first: Heather

last: O'Leary

first: Stephanie

last: Rellick

first: Debra

last: Piktel

first: Cheryl

last: Walton

last: Fortney

first: Stephen

last: Akers

first: Karen

last: Martin

first: Goran

last: Boskovic

first: Donald

last: Primerano

first: Laura

last: Gibson


title: Bifidobacterium bifidum BF-1 Suppresses Helicobacter pylori-Induced genes in Human Epithelial Cells

status: Public on Oct 13 2010

city: Kunitachi

first: Yukio

last: Shirasawa

address: 1796 Yaho

department: Central Institute for microbiological research

institute: Yakult Honsha Co., Ltd.

summary: We have shown that pre-incubation with Bifidobacterium bifidum (B. bifidum) strain BF-1, a probiotic strain known to improve H. pylori-associated gastritis, suppresses the induction of IL-8 by the pathogen.   To investigate how BF-1 affects gene expression in H. pylori-infected cells, we performed microarray analysis to assess gene expression in epithelial cells, which had been pre-incubated with BF-1 and infected with H. pylori.

overall_design: Four-condition experiment. Biological replicates with a dye-exchange experiment. Biological replicates with a longer incubation time experiment.

first: Fumiyasu

last: Ishikawa

first: Harue

last: Shibahara-Sone


title: Viral and transgene-free human induced pluripotent stem (iPS) cells using non-integrating Sendai virus (SeV) vectors.

status: Public on Sep 22 2010

city: Tsukuba

first: Noemi

last: Fusaki

state: Ibaraki

address: 6 Ohkubo

department: Cell Therapy & Regenerative Medicine

institute: DNAVEC Corporation

summary: Whole-genome expression analysis of non-integrating Sendai virus (SeV) vector and retroviral vector-generated human iPS cells.

overall_design: Parental fibroblast and induced pluripotent stem (iPS) cells.

first: Ban

last: Hiroshi

first: Mamoru

last: Hasegawa


title: Features of cryptic promoters and their varied reliance on bromodomain-containing factors

status: Public on Sep 23 2010

city: Kansas City

first: Samantha

middle: Gail

last: Pattenden

state: MO

address: 1000 E 50th Street

institute: Stowers Institute for Medical Research

summary:
The Set2-Rpd3S pathway is important for the control of transcription memory.  Mutation of components of this pathway results in cryptic transcription initiation within the coding region of approximately 30% of yeast genes. Specifically, deletion of the Set2 histone methyltransferase or Rco1, a component of the Rpd3S histone deacetylase complex leads to hyperacetylation of certain open reading frames (ORFs). We used this
mutant as a system to study the role of histone modifications and co-activator recruitment in preinitiation complex (PIC) formation. Specifically, we looked at the dependence of promoters on the bromodomain-containing RSC complex and the Bdf1 protein. We found that the dependence of cryptic promoters for these proteins varied. Overall, our data indicates that cryptic promoters are independently regulated, and their activation is dependent on factors that govern gene activation at canonical promoters.

overall_design: Two-sample experiment: chromatin immunoprecipitation (ChIP) sample vs. input. Biological replicates: 3 independent ChIP samples for each yeast strain.

middle: G

first: Madelaine

last: Gogol

first: Jerry

last: Workman


title: Human osteoblast treated with recombinant TGF-beta

first: James

last: Denvir

summary: Global expression profile of human osteoblast treated with recombinant TGF-beta compared to human osteoblast treated with growth media alone

overall_design: Dye-swap design with 6 biological replicates. Three arrays performed with TGF-beta treated samples on channel 1 and media-alone treated on channel 2; three arrays performed with TGF-beta treated samples on channel 2 and media-alone on channel 1.

first: Heather

last: O'Leary

first: Stephanie

middle: L

last: Rellick

last: Piktel


title: Transcription profiling of primed, ILT3-Fc treated CD8+ T suppressor cells vs. primed, untreated CD8+ counterparts

status: Public on Sep 02 2010

city: New York

first: Nicole

last: Suciu-Foca

state: NY

department: Pathology and Cell Biology

institute: Columbia University

summary:
CD3 T cells were first primed for 7 days with allogeneic APC in the presence of absence of ILT3-Fc, then were restimulated for 4 hours (with the original priming APC)
CD8 T cells were magnetically sorted from these cultures, and lysed for gene chip profiling.

overall_design: Two condition experiments, ILT3Fc treated vs untreated CD8+ T cells, (4) ILT3Fc treated replicates and (4) untreated replicates

first: George

last: Vlad

first: Chih-Chao

last: Chang


title: Human osteoblast treated with chemotherapy-treated bone marrow stromal cell conditioned media

summary: Global expression profile of human osteoblast treated with chemotherapy-treated bone marrow stromal cell conditioned media, compared to human osteoblast cells treated with diluent-control bone marrow stromal cell conditioned media. Goal is to identify genes regulated by chemotherapy in osteoblasts.

overall_design: Dye-swap design with 6 biological replicates. Three arrays performed with chemotherapy treated samples on channel 1 and diluent-control treated on channel 2; three arrays performed with chemotherapy treated samples on channel 2 and diluent-control on channel 1.

first: Debra

first: Cheryl

last: Walton

last: Fortney

first: Stephen

last: Akers

first: Karen

last: Martin

first: Goran

last: Boskovic

first: Donald

last: Primerano

middle: F

last: Gibson


title: Characterization of HT-29 cells cultured in NanoCulture plates

status: Public on Aug 25 2010

city: Eiheiji

first: Yukie

last: Yoshii

address: 23-3, Matsuoka　Shimoaizuki

department: Biomedical Imaging Research Center

institute: University of Fukui

summary: To characterize cells cultured in 3D culture system using NanoCulture plates (NCPs) (Scivax) and in 2D culture, we examined gene expression in HT-29 cells on day 1 or day 7 on NCPs or 2D monolayer culture, using DNA microarrays. From our study, tumour cells grown on NCPs were morphologically different from cells in 2D monolayers, and the cells on NCPs actively migrated and aggregated to form multicellular spheroids (MCSs). To characterize this cell migration and intercellular adhesion, we compared gene expression in HT-29 cells on day 1 on NCPs and 2D monolayer culture using DNA microarrays. We could not find any significant difference in gene expression related to cell migration or cell-cell adhesion between cells grown on NCPs and as 2D monolayers on day 1, showing that cells on NCPs migrated and aggregated without any changes in gene expression (3D Day1 vs 2D Day1 2Fold change.xls). We next characterized mature 3D MCSs on NanoCulture plates, by comparing the gene expression profile of MCSs on day 7 and day 1 (3D Day7 vs 3D Day 1 2Fold change.xls). On day 7, we found a statistically significant increase in the expression of genes related to multicellular organization, intercellular signalling and the response to hypoxia. DNA microarray analysis also showed that the expression of genes that have been reported to be target genes for the transcription factor hypoxia-inducible factor 1 (HIF-1) (Ke, Q. & Costa, M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 70, 1469-1480 (2006); Semenza, G.L. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29, 625-634.) was increased on day 7. When compared day 7 with 2D culture cells, the same tendency was observed (3D Day7 vs 2D Day 7 2Fold change.xls).

overall_design: HT-29 cells were maintained routinely as 2D monolayers in a humidified atmosphere of 5% CO2 in air at 37 °C in the growth medium recommended by suppliers. Exponentially growing cells were used in experiments, after trypsinization to detach them from the plates. Viable cells were counted using trypan blue dye-exclusion. For experiments, cells were seeded in 96-well plates at 1×10^4 cells/100 ul of NanoCulture Medium-M (Scivax) in either NCPs (Scivax), or in polystyrene plates for 2D monolayers, for 1 day or 7 days. Cells were collected and used for RNA extraction and DNA microarray analysis.

first: Atsuo

last: Waki


title: Gene expression analysis of Human CD34+ cells derived from bone marrow, mobilized peripheral blood, umbillial cord blood using Agilent Whole Human Genome Oligo Microarrays (one-color)

status: Public on Sep 20 2010

city: Indianapolis

first: Brahmananda

middle: Reddy

last: Chitteti

state: IN

address: 980 W Walnut Streeet R3-c356

institute: Indiana University School of Medicine

summary: In order to identify genes associated with the engraftment potential of human hematopoietic stem cells, we have employed whole genome microarray expression profiling of G0 and G1 phase CD34+ cells derived from bone marrow, mobilized peripheral blood, and umbilical cord blood. Samples were collected from healthy adult volunteers after obtaining informed consent according to the guidelines of the Investigational Review Board of Indiana University School of Medicine. CD34+ cells were selected and fractionated into G0 and G1 phases of cell cycle  on a flow cytometer. Purity of sorted cells was further confirmed by qRT-PCR by measuring the relative expression of Ki67. Sorted cells were subjeccted to microarray analysis.

overall_design: Three biological replicates of sorted and confirmed G0 and G1 cells from bone marrow, mobilized peripheral blood, and umbilical cord blood (total of eighteen samples) were subjected to microarray analysis. To generate distinct and unique sets of data, we did not pool multiple samples from any tissue studied so that each sample or its replicate was from a single donor.

first: Yunong

last: Liu

first: Edward

last: Srour


title: Changes in polysome loading as a consequence of RHA downregulation [Agilent]

status: Public on Aug 19 2010

city: Columbus

first: Kathleen

last: Boris-Lawrie

state: OH

address: 1925 Coffey Rd

department: Department of Veterinary Biosciences

institute: The Ohio State University

summary: RNA helicase A (RHA) binds its target transcripts at the post-transcriptional control element (PCE) located in the 5’ untranslated region (UTR). This interaction represents an “RNA switch” that regulates protein synthesis. Down regulation of RHA by siRNAs was used to identify transcripts with RHA-dependent translation. Reduced accumulation of RNA in polysomes was monitored with microarrays.

overall_design: Cytoplasmic lysates of cells treated with RHA targeted or non-silencing control siRNAs were separated by sucrose density gradient centrifugation. Ribosomal RNA profiles were generated, containing heavy polysomes were collected, and RNA was extracted.

first: Julia

last: Hernandez


title: Gene expression analysis of transcriptome changes regulated by RANKL-RANK signaling in macrophage cell line RAW 264.7

status: Public on Aug 24 2010

city: Boston

first: Parul

last: Sharma

state: MA

address: 320 Longwood Ave

department: Orthopedic Surgery

institute: Children's Hospital, Boston

summary:
Bone remodeling relies on the precise developmental control of osteoclasts that resorb bone and osteoblasts that lay down fresh bone matrix. The hematopoietically derived osteoclast follow a tigthly controlled genetic program where specific genes are up-regulated to aid bone resorption.Some of them for instance control membrane polarization, others transporters and pumps  to move the acid and proteases intot he resorption pit.
We used microarrays to detail the global programme of gene expression underlying differentiation of osteoclasts and identified distinct classes of up-regulated genes during this process.

overall_design: RAW cells were selected at different time periods of culture during the osteoclast differentiation process for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain more homogenous temporal resolution of expression profiles by using a cell line RAW 264.7 and including data of control cells that had been cultured for similar time periods without RANKL.

first: Peter

middle: V

last: Hauschka

first: Somying

last: Patntirapong

first: Steven

last: Hann


title: RNA immunoprecipitation to identify RHA-binding transcripts in HEK293 cells

summary: Given that RHA regulates translation by binding a PCE located at the 5’ UTR of the target transcripts a genome-wide screen was performed to identify mRNAs that bind RHA in vivo. The results from four experiments with samples obtained in four independent RNA immunoprecipitations identified 375 transcripts that co-immunoprecipitate with FLAG RHA.

overall_design: Since N-terminal FLAG tagged RHA specifically co-immunoprecipitates PCE-containing mRNAs HEK293 cells were transfected with a CMV-FLAG-RHA construct. Cytoplasmic lysates were  immunoprecipitated with anti-FLAG beads. RNA was extracted from the immunoprecipitate and used to probe a human Agilent expression arrays. An immunoprecipitation with cells transfected with empty FLAG plasmid was used as negative control.


title: Identification of RNA helicase A translation target mRNAs at the intersection of RNA precipitation and polysome arrays

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

sample_organism:
Chlorocebus aethiops
Homo sapiens

overall_design: Refer to individual Series


title: Identification of genes differentially expressed between control and EHF siRNA transfected HCT116 cells

status: Public on Jun 29 2010

city: Tokyo

first: Tetsu

last: Akiyama

address: 1-1-1, Yayoi, Bunkyo-ku

department: Institute of Molecular and Cellular Biosciences

institute: Tokyo University

summary: we found that EHF allowed colon tumor cells to escape p53-mediated apoptosis.We performed microarray analysis to find the target genes of EHF.

overall_design: A colorecal cancer cell line HCT116 was transfected with an siRNA targeting EHF or negative control.

first: Kenzui

last: Taniue

first: Tomoatsu

last: Hayashi


title: Human CEM Cells: Control vs. 2h or 4.5h Cluvenone Treated

status: Public on Aug 11 2010

city: Columbia

first: Diego

last: Altomare

state: SC

address: 715 Sumter Street, Room 617

department: Department of Drug Discovery and Biomedical Sciences

institute: University of South Carolina

summary: Transcriptional profiling of human CEM cells comparing control untreated CEM cells with either 2h or 4.5h cluvenone-treated CEM cells.

overall_design: Two-timepoint experiment, CEM vs. Cluvenone-Treated CEM (2h and 4.5h). Biological replicates: 4 control, 4 2h-treated, 4 4.5h-treated, independently grown and harvested. Pooled controls. One replicate per array.

first: Ayse

last: Batova

first: Kim

last: Creek


title: Microarray analysis of therapeutic (GM4) and non therapeutic (GM) NOD DC

status: Public on Jun 24 2010

city: Pittsburgh

first: Penelope

middle: Anne

last: Morel

state: PA

address: 200 Lothrop Street, BST E1048

department: Immunology

institute: University of Pittsburgh

summary: We have previously demonstrated that bone marrow-derived DC can prevent diabetes development and halt progression of insulitis in NOD mice, the mouse model of type 1 diabetes (T1D). The DC population that was most effective in this therapy had a mature phenotype, expressed high levels of costimulatory molecules and secreted low levels of IL-12p70. The protective DC therapy induced regulatory Th2 cells that shifted the dominant Th1 environment, present in NOD mice, to a mixed Th1/Th2 milieu.  Microarray analysis of therapeutic and non-therapeutic DC populations revealed several novel molecules that could play important roles in the observed DC-mediated therapy. The therapeutic DC population expressed a unique pattern of costimulatory molecules and chemokines, which were confirmed by flow cytometry and ELISA assays. We have performed in vitro chemotaxis assays that demonstrated the therapeutic DC preferentially attracted Th2 cells, as compared to Th1, Treg or naïve T cells. In addition we quantified the in vivo migration of activated islet-specific T cells to the pancreas using novel cell labeling techniques and 19F nuclear magnetic resonance. A subcutaenous injection of therapeutic DC alters the migration of both Th1 and Th2 cells to the pancreas, and Th1 cells appeared in the lymph node draining the site of DC injection. These results suggest that the therapeutic function of DC is mediated in part by the chemoattractive properties of these DC for diabetogenic Th1 cells.

overall_design:
Bone marrow cells from NOD mice were cultured in GM or GM + IL-4 to generate DC. DC were purified on metrizamide gradients after 4 days of culture. Purified DC were stimulated with LPS + IFNg and RNA taken 0, 6 and 20 hours after stimulation. Three biological replicates were performed but not all RNA samples were good.
The are 3 replicates for GM/0, GM4/0, GM4/6 and 2 replicates for GM/6, GM/20 and GM4/20

first: Maryam

last: Feili-Hariri

first: Rajan

last: Munshi

first: Ivet

last: Bahar


title: Molecular characterization of head and neck cancer cell model genomes

status: Public on Jun 26 2010

city: Vancouver

first: Ivy

last: Tsui

state: BC

address: 675 West 10th Avenue

department: Cancer Genetics and Developmental Biology

institute: BC Cancer Research Centre

type: Genome variation profiling by genome tiling array

summary:
To provide full characterization of genome changes in six commonly used head and neck cancer cell lines.
These data will serve as an excellent resource when designing future experiments that attempt to model HNSCC behaviour.

overall_design: Six commonly used ATCC head and neck cancer cell lines are analyzed.

first: Cathie

last: Garnis


title: Identification of cis- and trans-acting factors involved in the localization of MALAT-1 to nuclear speckles

status: Public on Jun 02 2010

first: RANDEEP

last: RAKWAL

address: 1-1-1 Tennodai

department: Institute of Health and Sport Sciences

institute: University of Tsukuba

summary: MALAT-1 is a long mammalian non-coding transcript of approximately 8500 nucleotides.  It is localized to nuclear speckles despite its mRNA-like characteristics, which usually result in the export of transcripts to the cytoplasm.  In the present study, we report the identification of several factors that influence the nuclear speckle localization of MALAT-1, and we provide evidence that MALAT-1 is involved in the regulation of gene expression.  Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm, but is stably retained within the nucleus.  Analysis of MALAT-1 fragments showed that MALAT-1 contains two distinct nuclear speckle-directing elements (between nucleotides 1961 to 3040 and 6008 to 7011 of the MALAT-1 sequence).  The knockdown of the nuclear speckle proteins, RNPS1, SRm160 or IBP160, resulted in the diffusion of MALAT-1 to the nucleoplasm.  In addition, we have demonstrated that depletion of MALAT-1 represses the expression of several genes.  This repression of gene expression also occurs in response to the delocalization of MALAT-1 from the nuclear speckles.  These results suggest that RNPS1, SRm160 and IBP160 contribute to the localization of MALAT-1 to nuclear speckles where it is involved in regulating gene expression

overall_design: We used DNA microarray analysis to analyze differentially expressed (up- and down-regulated) genes in cells depleted of MALAT-1 by RNAi.  Microarray analysis using the Agilent Whole Human Genome 4 x 44K oligo microarray in two independent MALAT-1 knockdown cell lines transfected with different siRNAs. Total RNA (800 ng) was labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent).  Fluorescently labeled cRNAs, derived from total RNA isolated from control cells, and from MALAT-1 knockdown cells, were hybridized to the same microarray slide carrying 60-mer probes from the whole human genome 4 x 44K oligo microarray kit (Agilent Technologies, Palo Alto, CA, USA).  A flip labeling (dye-swap or reverse labeling with Cy3 and Cy5 dyes) procedure was followed to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cDNA.

first: Ryu

last: Miyagawa

first: Rie

last: Mizuno

first: Yo

last: Nakamura

first: Kenichi

last: Ijiri

first: Randeep

last: Rakwal

first: Junko

last: Shibato

first: Yoshinori

last: Masuo

first: Tetsuro

last: Hirose

first: Nobuyoshi

last: Akimitsu


title: Identification and validation of NOL5A and RPS2 as potential therapeutic targtes in colorectal cancer

status: Public on Jun 01 2010

city: Goettingen

first: Marian

last: Grade

address: Robert-Koch-Str. 40

department: Surgery

institute: University Medical Center Goettingen, Georg-August-University

summary:
Identification and validation of NOL5A and RPS2 as potential therapeutic targets in colorectal cancer using a functional genomics approach.
To identify potential therapeutic targets for colorectal cancer, we first assessed the functional and molecular consequences of RNAi mediated silencing of candidate genes derived from previously performed gene expression analyses. We then generated gene expression signatures after RNAi against HMGA1, RRM2, TACSTD2, RPS2, and NOL5A.

overall_design: To assess the consequences of silencing specific genes on global gene expression levels, triplicate transfections were independently performed. Cells for lyzed 48 hours or 72 hours after transfection (depending on the target gene), and total RNA was isolated for each transfection. For each sample, one array experiment was subsequently performed.

first: Tim

last: Beissbarth

first: Natasha

last: Caplen

first: Thomas

last: Ried


title: A murine-ES like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

status: Public on Jun 07 2010

first: Niels

last: Geijsen

address: 185 Cambridge St. CPZN 4-4256

department: Center for Regenerative Medicine

institute: Massachusetts General Hospital

summary: To analyze the gene expression profiles of different states of human pluripotent stem cells

overall_design: The total gene expression profiles of human embryonic stem cells, human induced pluripotent stem cells and human LR5-iPS cells were analyzed by Agilent Human Whole Genome one-color microarray.

first: Christa

last: Beucker

first: Hsu-Hsin

last: Chen

first: Jose

last: Polo

first: Laurence

last: Daheron

first: Lei

last: Bu

first: Joost

last: Gribnau

first: Stefan

last: Barakat

first: Patricia

last: Okwieka

first: Konrad

last: Hoechedlinger


title: Genome-Wide Expression Profiling Deciphers Host Responses Altered during Dengue Shock Syndrome and Reveals the Role of Innate Immunity in Severe Dengue

status: Public on Jul 21 2010

city: Marseille cedex 07

first: Stephanie

last: Devignot

address: Parc du Pharo BP60109

department: Virology

institute: IMTSSA

summary:
Background
Deciphering host responses contributing to dengue shock syndrome (DSS), the life-threatening form of acute viral dengue infections, is required to improve both the differential prognosis and the treatments provided to DSS patients, a challenge for clinicians.

Methodology/Principal Findings
Based on a prospective study, we analyzed the genome-wide expression profiles of whole blood cells from 48 matched Cambodian children: 19 progressed to DSS while 16 and 13 presented respectively classical dengue fever (DF) or dengue hemorrhagic fever grades I/II (DHF). Using multi-way analysis of variance (ANOVA) and adjustment of p-values to control the False Discovery Rate (FDR<10%), we identified a signature of 2959 genes differentiating DSS patients from both DF and DHF, and showed a strong association of this DSS-gene signature with the dengue disease phenotype. Using a combined approach to analyse the molecular patterns associated with the DSS-gene signature, we provide an integrative overview of the transcriptional responses altered in DSS children. In particular, we show that the transcriptome of DSS children blood cells is characterized by a decreased abundance of transcripts related to T and NK lymphocyte responses and by an increased abundance of anti-inflammatory and repair/remodeling transcripts. We also show that unexpected pro-inflammatory gene patterns at the interface between innate immunity, inflammation and host lipid metabolism, known to play pathogenic roles in acute and chronic inflammatory diseases associated with systemic vascular dysfunction, are transcriptionnally active in the blood cells of DSS children.

Conclusions/Significance
We provide a global while non exhaustive overview of the molecular mechanisms altered in of DSS children and suggest how they may interact to lead to final vascular homeostasis breakdown. We suggest that some mechanisms identified should be considered putative therapeutic targets or biomarkers of progression to DSS.

overall_design: Whole blood genome-wide expression profiles of Cambodian children (3-15 year old) infected with dengue virus, having different clinical outcomes were compared. The studied cohort included 16 acute dengue fever samples, 13 acute dengue hemorrhagic fever samples and 19 acute dengue shock syndrome samples, classified according to the 1997 WHO criteria and randomised for age, gender, viral serotype and day of blood sampling after onset of fever. Microarray data were normalised using the quantile method. Multi-way ANOVA was used to compared the three clinical groups, at several False Discovery Rate of 10. Unsupervised Hierarchical Clustering (TreeView) showed that DSS patients clustered together (17 out of 19), identifying a gene-signature of DSS. Bio-informatics-based analysis using the demonstration version 7.1 of Ingenuity Pathway Analysis software (IPA; Ingenuity Systems, www.ingenuity.com) associated with manual and litterature-based analysis was carried out to identify the most relevant functional processes associated with the identified DSS gene expression profile. This was done by combining most informative canonical pathways identified using IPA, genes having the strongest association with the disease phenotype based on ANOVA analysis, and similarities to molecular patterns altered in other systemic inflammatory processes associated with endothelial dysfunction.

first: Cedric

last: Sapet

first: Veasna

last: Duong

first: Aurelie

last: Bergon

first: Pascal

last: Rihet

first: Sivuth

last: Ong

first: Patrich

last: Lorn

first: Norith

last: Chroeung

first: Sina

last: Ngeav

first: Hugues

middle: J

last: Tolou

first: Philippe

last: Buchy

last: Couissinier-Paris


title: Direct generation of induced pluripotent stem cells from human non-mobilized blood

city: Machida-shi

first: Kenji

last: Nagao

address: 3-6-6, Asahi-machi,

department: Innovative Drug Research Laboratories

institute: Kyowa Hakko Kirin Co., Ltd.

summary: The use of induced pluripotent stem cells (iPSCs) is an exciting frontier in the study and treatment of human diseases through the generation of specific cell types. Here we show the derivation of iPSCs from human non-mobilized peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) by retroviral transduction of OCT3/4, SOX2, KLF4 and c-MYC. The PB- and BM-derived iPSCs were indistinguishable from human embryonic stem cells (hESCs) with respect to morphology, expression of surface antigens and pluripotency-associated transcription factors, global gene expression profiles and differentiation potential in vitro and in vivo. Infected PB and BM MNCs gave rise to iPSCs in the presence of several cytokines, although transduction efficiencies were not high. We found that 5×105 PB MNCs, which corresponds to less than 1 ml of PB, was enough for the generation of several iPSC colonies. Generation of iPSCs from MNCs of non-mobilized PB, with its relative efficiency and ease of harvesting, could enable the therapeutic use of patient-specific pluripotent stem cells.

overall_design: Gene expression profiling was performed in human BMMNCs, PBMNCs, hBMMNC derived iPS cell lines and hPBMNC derived iPS cell lines.

first: Atsushi

last: Kunisato

first: Mariko

last: Wakatsuki

first: Haruna

last: Shinba

first: Toshio

last: Ota

first: Isao

last: Ishida


title: RNA Polymerase mapping during stress responses reveals widespread nonproductive transcription in yeast

status: Public on Jul 16 2010

city: Worcester

first: Oliver

last: Rando

address: 364 Plantation St.

institute: UMass Medical School

summary: The use of genome-wide RNA abundance profiling by microarrays and deep sequencing has spurred a revolution in our understanding of transcriptional control. However, changes in mRNA abundance reflect the combined effect of changes in RNA production, processing, and degradation, and thus, mRNA levels provide an occluded view of transcriptional regulation. To partially disentangle these issues, we carry out genome-wide RNA Polymerase II (“Pol2”) localization profiling in budding yeast in two different stress response time courses. While mRNA changes largely reflect changes in transcription, there remains a great deal of variation in mRNA levels that is not accounted for by changes in Pol2 abundance. We find that genes exhibiting “excess” mRNA produced per Pol2 are enriched for those with overlapping cryptic transcripts, indicating a pervasive role for nonproductive or regulatory transcription in control of gene expression. Finally, we characterize changes in Pol2 localization when Pol2 is genetically inactivated using the rpb1-1 temperature-sensitive mutation. We find that Pol2 is lost from chromatin after roughly an hour at the restrictive temperature, and that there is a great deal of variability in the rate of Pol2 loss at different loci. Together, these results provide a global perspective on the relationship between Pol2 and mRNA production in budding yeast.

overall_design: The array studies consisted of 4 time courses in which the genome-wide location of RNA Polymerase II was mapped via chromatin immunoprecipitation (ChIP) in BY4741 S. cerevisiae cells or in such cells bearing the temperature sensitive mutation rpb1, in response to heat shock at 37 degrees C and/or 1.5 mM diamide.  All samples were hybridized as the ChIP sample (Cy3 labeled, channel 1) versus its cognate ChIP input sample (Cy5 labeled, channel 2).  The first time course was a heat shock in wild-type BY4741 cells.  The second time course was a heat shock in BY4741 rpd1 cells.  The third time course was a diamide treatment of BY4741 rpd1 cells.  The fourth time course was a 10 minute heat shock in BY4741 rpd1 cells followed by treatment from 15-60 minutes with diamide (3 samples).  In the first three time courses the time range is 0-120 minutes (5 samples).  A total of 18 arrays were used in this study.  No additional replicates or dye-flip experiments were performed.

first: Tae

last: Kim

first: Chih

last: Liu

first: Moran

last: Yassour

first: John

last: Holik

first: Nir

last: Friedman

first: Stephen

last: Buratowski


status: Public on Aug 03 2010

city: Sharnbrook

first: Fei Ling

last: Lim

state: Bedfordshire

address: Colworth Science Park

country: United Kingdom

institute: Unilever

summary: Gene expression changes in Peripheral Blood Mononuclear cells (PBMC) induced by physical activity was investigated in  sedentary middle-aged men (mean age 52.6 years and BMI 29.1) who undertook a 24-week physical activity programme with blood sampling in the pre-exercise period ,  at the end of 24-weeks prescribed physical activity , and following a two-week detraining period.

overall_design: AgilentTM Whole Human Genome Oligo Microarrays were utilised to examine the effects of physical activity on mRNA expression profiles of the Peripheral Blood Mononuclear cells (PBMC) at 3 time points (pre-exercise, after 24 weeks physical activity, and at 26 weeks after 2 weeks detraining. There were 12 participants in this programme.

first: Dawn

last: Mazzatti

first: Daniella

last: Markovitch

first: FeiLing

first: Sarah

last: Askew

first: Tina

last: Hurst

first: Dylan

last: Thompson


title: Adrenal gland gene expression from adult (12-week old) hypertensive BPH and adult (12-week old) hypotensive BPL mice

status: Public on Jun 08 2010

city: La Jolla

first: Ryan

last: Friese

state: CA

institute: University of California San Diego

summary: The objective of the experiment is to determine the genes differentially expressed in the adrenal gland of adult (12-week) BPH and BPL mice.

overall_design: Adrenal glands from adult (12-week) hypertensive BPH mice (n=3) and adult (12-week) hypotensive BPL mice (n=3).


title: Comparison of expression profiles of once and three time linearly amplified samples

status: Public on May 20 2010

city: Leipzig

first: Thomas

last: Giger

address: Deutscher Platz 6

department: Evolutionary Genetics

institute: Max-Planck-Institute of Evolutionary Anthropology

summary: In order to assess the impact of three rounds of linear amplification on the technical reproducibility of gene expression measurements, we performed twelve microarray experiments. We analysed mouse RNA from cortex, cerebellum and liver from one individual. One RNA sample of 5µg from each of the three different tissues was processed according to the standard Affymetrix protocol and hybridized onto mouse gene expression arrays MG_U74Av2. Three additional samples from each tissue of 1ng were processed according to a modified procedure that involves three linear amplifications before hybridization onto the microarray chips.

overall_design: One mouse was analysed. One RNA sample of 5µg from each of liver, cortex, and cerebellum were processed according to the standard Affymetrix protocol and hybridized onto mouse gene expression arrays MG_U74Av2. Three additional samples (technical replicates) from each tissue of 1ng were processed according to a modified procedure that involves three linear amplifications before hybridization onto the microarray chips, resulting in 12 microarray experiments.

first: Philipp

last: Khaitovich

first: Mehmet

last: Somel

first: Anna

last: Lorenc

first: Esther

last: Lizano

first: Laura

last: Harris

first: Margaret

last: Ryan

first: Martin

last: Lan

first: Matthew

last: Wayland

first: Sabine

last: Bahn

first: Svante

last: Pääbo


title: Transcriptome analysis of highly metastatic cell line

city: Nagaizumi-cho

first: Keiichi

last: HATAKEYAMA

state: Sunto-gun, Shizuoka

address: 1007 Shimonagakubo

institute: Shizuoka Cancer Center Research Institute

summary: A prediction of peritoneal recurrence is of significance using metastasis-related biomarker. This work describes a combined analysis of proteome and transcriptome data for biomarker discovery in highly metastatic cell line. We used nano-flow liquid chromatography (LC) linear ion trap time-of-flight mass spectrometry (LIT-TOF MS) and cDNA microarray to identify specific protein differentially expressed between a highly metastatic stomach cancer cell line MKN-45-P and its parental cell line MKN-45. In total, 240 proteins were found to be expressed between the two cell lines. Of these, 75 proteins (31%) and 49 proteins (20%) were only identified from MKN-45-P and MKN-45 respectively. An mRNA expression of 1533 genes was up-regulated in MKN-45-P compared with MKN-45. No close correlation was found between proteomic and transcriptomic analysis. Interestingly, 4 of 240 proteins (1.6%) were involved in the up-regulated mRNA expression.

overall_design: Comparison analysis of gene expression between highly metastatic gastric cancer cell line MKN-45-P and its parental cell line MKN-45.

last: Ohshima

last: Hatakeyama


title: Effects of retinoids on estrogen-receptor-positive and -negative breast carcinoma cells:  mRNA profiling

status: Public on May 21 2010

city: Milano

first: Maddalena

last: Fratelli

address: Via La Masa, 19

institute: "Mario Negri" Institute

summary:
The overall study explores differential sensitivity of estrogen-receptor-positive and -negative breast carcinoma cells to retinoids via gene expression and microRNA profiling in MCF7 and MDA-MB-231 cells.
This Series reports results of transcriptional profiling of breast carcinoma cell lines comparing the effects of retinoic acid treatment (6 and 48 hours) on estrogen-receptor-positive (MCF7) and estrogen-receptor-negative (MDA-MB-231) cells.

overall_design: mRNA profiling: Retinoic-acid-treated (1microM) vs vehicle-treated cells, two time points (6 and 48h), two cell lines (MCF7 and MDA-MB-231). Two biological replicates for each condition, balanced dye design.

first: Mineko

last: Terao

last: Tsykin

first: Gregory

last: Goodall

first: Enrico

last: Garattini


title: Effects of retinoids on estrogen-receptor-positive and -negative breast carcinoma cells: mRNA and miRNA profiling

type:
Expression profiling by array
Non-coding RNA profiling by array

platform_organism:
Arabidopsis thaliana
Saccharomyces cerevisiae
Caenorhabditis elegans
Drosophila melanogaster
Gallus gallus
Macaca mulatta
Homo sapiens
Bos taurus
Mus musculus
Rattus norvegicus


title: Effect of IL6 level on gene expression changes in Peripheral Blood Mononuclear cells (PBMC) induced by physical activity

first: Fei Ling

last: Lim

summary: A number of chronic, age-related diseases are associated with elevated markers of inflammation such as interleukin-6 (IL-6). In this study, we investigated the hypothesis that sedentary individuals with disparate basal serum IL-6 respond differentially to a structured physical activity programme. Gene expression changes in Peripheral Blood Mononuclear cells (PBMC) induced by physical activity was investigated in  sedentary, middle-aged men (mean age 52.6 years and BMI 29.1), with relatively high or low basal serum IL-6 levels (mean of 2.13 and 0.59pg/ml respectively), who undertook a 24-week physical activity programme with blood sampling in the pre-exercise period and at the end of 24-weeks prescribed physical activity.

overall_design: AgilentTM Whole Human Genome Oligo Microarrays were utilised to examine the effects of physical activity on mRNA expression profiles of the Peripheral Blood Mononuclear cells (PBMC) at 2 time points (pre-exercise, and after 24 weeks physical activity. There were 6 individuals per group.

first: Dawn

last: Mazzatti

first: Daniella

last: Markovitch

first: FeiLing

first: Sarah

last: Askew

first: Tina

last: Hurst

first: Dylan

last: Thompson


title: CpG islands recruit a histone H3 lysine 36 demethylase [Agilent data]

status: Public on Apr 27 2010

first: Peter

last: Park

address: 10 Shattuck St

department: Center for Biomedical Informatics

institute: Harvard Medical School

summary: Transcription factors that bind small DNA motifs embedded in promoters play a central role in controlling gene expression. However, in addition to these elements, up to 70% of genes in higher eukaryotes also have high levels of non-methylated cytosine/guanine base pairs (CpGs) surrounding promoters and gene regulatory units. These features, called CpG islands, were identified over twenty years ago but there remains little mechanistic evidence to suggest how these enigmatic elements contribute to promoter function, with the exception that they are refractory to epigenetic silencing by DNA methylation. Here we show that CpG islands directly recruit the H3K36 specific lysine demethylase enzyme KDM2A. Genome wide analyses by ChIP-seq demonstrated a striking global association of KDM2A with CpG islands. Nucleation of KDM2A at these elements resulted in removal of H3K36 methylation creating CpG island chromatin that is uniquely depleted of this modification. KDM2A utilizes a zinc finger CxxC (ZF-CxxC) domain that specifically recognizes non-methylated CpG DNA and binding is blocked when the CpG DNA is methylated, thus constraining KDM2A to nonmethylated CpG islands. These data expose a remarkably straightforward mechanism through which KDM2A delineates a unique architecture that differentiates CpG island chromatin from bulk chromatin.

overall_design: Two cell lines were used in this study: a control line (LMP) with wildtype levels of KDM2A, and a KDM2A knockdown line (RNAi) in which KDM2A levels were depleted by approximately 60% using an shRNA-based approach. For each cell line, RNA was extracted from cells collected on two different dates (rep1 and rep2).

first: Neil

middle: P

last: Blackledge

first: Jin

last: Zhou

first: Michael

middle: Y

last: Tolstorukov

first: Robert

last: Klose


title: Wild-type and COUP-TFI-/- newborn inner ear microarrays

status: Public on May 31 2010

city: Houston

first: Fred

middle: A.

last: Pereira

address: One Baylor Plaza

department: Hufftington Center on Aging

institute: Baylor College of Medicine

summary: In order to establish a list of candidate direct COUP-TFI gene targets in the inner ear, we analyzed the differential gene expression profiles of the wild-type and the COUP-TFI–/– P0 inner ears.

overall_design: We performed a total of 8 microarray experiments using Affymetrix MG-U74Av2 chips, including 2 biological and 2 experimental replicates per genotype sample.  Due to limiting RNA yields from the newborn inner ear, each microarray chip was hybridized with an RNA pool from multiple tissue samples. We analyzed our data using two different algorithms: GC-Robust Multi-Array (GCRMA) and dChip.  Each normalized expression dataset was subsequently analyzed by 2-way ANOVA, evaluating both genotype and experimental effects.  This statistical approach allowed us to 1) account for an experimental effect observed in the expression value of many genes, therefore increasing the power of the analysis, and 2) filter out potential expression differences due to contamination during dissections (contaminating genes would present as probes with a significant interaction p-value).  Using this methodology, the gene hits from the GCRMA-normalized expression dataset consisted of 256 genes with a significant genotype effect (p<0.01) and no interaction (p>0.01).  Similar cutoffs applied on the dChip-normalized dataset resulted in 250 significant gene hits.  Within both groups, COUP-TFI has the lowest genotype p-value, validating our statistical approach.

first: Celina

last: Montemayor

first: Feng

last: Lin

first: Scott

middle: D

last: Pletcher

middle: A


title: Global analysis of differential polyadenylation of mRNAs in CPEB1 knockdown and CPEB4 knockdown at G2/M.

status: Public on Apr 01 2010

city: Barcelona

first: Isabel

last: Novoa

address: Dr. Aiguader, 88

department: Gene Regulation

institute: Center for Genomic Regulation

summary:
We compared the poly(A) tail length status of mRNAs of HeLa cells expressing a CPEB1 shRNA (CPEB1 knockdown) versus a control shRNA, and expressing a CPEB4 shRNA (CPEB4 knockdown) versus a control shRNA. Results provide insight into the extent of gene regulation mediated by CPEB1 and CPEB4 activity during mitotic cell cycle progression.  The different shRNA expressing cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine),  and samples were taken after 8 hours release (G2/M phase). For each shRNA expressing HeLa cell line total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 μg of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ºC to isolate mRNAs with short poly(A) tail (<30As, SHORT fraction). Oligo(dT) chromatography: mRNAs were purified independently of their poly(A) tail length with Ambion Poly(A)Purist kit from 20 μg total RNA (ALL fraction).  Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261.

Keywords: knock-down experiment

overall_design: Comparison of ALL fraction mRNAs and SHORT fraction mRNAs of cells expressing control shRNA, or CPEB1 shRNA, or CPEB4 shRNA measured after 8 hours release (G2/M) from double thymidine blockade; hybridisations are then used for comparison between control shRNA expressing cells and each of the two CPEB1 shRNA or CPEB4 shRNA expressing cells, as well as, comparison between the two knockdowns; 3 biological replicates for each shRNA expressing cells; two technical replicates with dye swapping per comparison.

first: Javier

last: Gallego

first: Raul

last: Mendez


title: Gene expression changes in PBMCs from metabolic syndrome patients after acute intake of phenol-rich virgin olive oil

status: Public on Apr 20 2010

city: New York

first: Juan

last: Ruano

address: 1230 York ave

institute: Rockefeller University

summary: Some studies have shown that acute intake of high-phenol virgin olive oil reduces pro-inflammatory, pro-oxidant and pro-thrombotic states, but nowadays still remains unclear if those effects attributed to its phenolic fraction could be exerted at transcriptional level in vivo. Two virgin olive oil-based breakfasts with high (398 ppm) and low (70 ppm) content of phenolic compounds were administered to twenty patients (9 men and 11 women) with metabolic syndrome following a double-blinded random crossover design. Prior to the first breakfast intervention, participants followed a 6-week washout period in which they were instructed to not take vitamins, soy supplements, or any drug. To eliminate the potential effect that might exist in their usual dietary habits, all subjects followed a low-fat, carbohydrate rich (CHO) diet during this period and untill the end of the study. In the 24 h before each breakfast intervention, participants were instructed to avoid consuming phenol-rich foods such as fruit or juices, wine, grape juice, chocolate, coffee, tea, olive oil, or soya, and to refrain from intense physical exercise during that period. After a 12 h fasting and following a randomized sequential crossover design with one-week washout period, participants reported to the hospital and received two fat meals consisting of 60 g of white bread, 40 mL of VOO (CANOLIVA®, Antonio Cano e Hijos™, Córdoba, Spain) with high (398 ppm) or low (70 ppm) content in phenolic compounds, and 60,000 IU of vitamin A per m2 of body surface. Olive oil with low content in phenolic compounds was obtained as a result of extraction by physical procedures of most of the phenolic compounds in the high-phenol olive oil, so that both oils kept a similar composition of the remaining macro- and micronutrients. Throughout the 4-h duration of the study, the subjects performed no physical activity, nor did they consume anything but water. Gene expression was performed on peripheral blood mononuclear cells (PBMCs) at postprandial period (4 hours after breakfast) by microarray analysis and verified by qRT-PCR.

overall_design: Two sets of dye-swaped experiments were performed making a total of four technical replicates per subject. Each of the hybridizations compared total RNA from PBMCs obtained 4h after the intake of virgin olive oil with high phenolic content vs. total RNA from PBMCs obtained 4h after low-phenol virgin olive oil consumption.

first: A

last: Camargo

first: J

middle: M

last: Fernandez

first: L

last: Parnell

last: Jimenez

first: M

last: Santos-Gonzalez

first: C

last: Marin

first: P

last: Perez-Martinez

last: Uceda

last: Lopez-Miranda

first: F

last: Perez-Jimenez


title: An Integrated Network of Androgen Receptor and TMPRSS2-ERG Gene Fusion in Prostate Cancer Progression (I)

status: Public on May 18 2010

city: Chicago

first: Jindan

last: Yu

state: IL

address: 303 E. Superior St.

department: Medicine - Hem/Onc

institute: Northwestern University

summary:
Androgen receptor (AR) is a transcription factor that plays a central role in the growth and development of the normal prostate and its malignant transformation.  More recently, a majority of prostate cancers have been shown to harbor recurrent gene fusions of the androgen-regulated gene, TMPRSS2, to the oncogenic ETS transcription factor ERG.  Here we employed chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-Seq) to explore the genome-wide localization of these transcription factors in human prostate cancer cell lines as well as tissues.  Unexpectedly, transcriptional networks emanating from AR and ERG were found to be highly overlapping.  Furthermore, AR was found to regulate known 5’ fusion partners in prostate cancer including TMPRSS2, as well as negatively regulating its own expression.  While induced by androgen through fusion to TMPRSS2, ERG itself was shown to inhibit AR expression and positively regulate the genomic locus of wild-type ERG, thus revealing multiple levels of molecular cross-talk between AR and ERG.  Importantly, androgen-sensitive prostate cancer cells in which ERG is overexpressed are able to proliferate and invade in the absence of androgen.  Thus, we dissected the intertwined genomic landscape of two master transcriptional regulators of prostate cancer and suggest a role for ERG in maintaining transcriptional networks necessary for androgen-independent prostate cancer growth.  These studies may suggest that future therapies against prostate cancer should target both AR and ERG, rather than AR alone, in order to achieve maximum effectiveness.

Keywords: Genetic modification

overall_design: time-course analysis of androgen treatment in LNCaP cells, and analysis of ERG overexpression and androgen effect in VCaP cells

first: Arul

last: Chinnaiyan


title: Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations

status: Public on Mar 19 2010

city: Cambridge

first: Albert

middle: W

last: Cheng

address: Room 453, 9 Cambridge Center

institute: Whitehead Institute

summary: The presence of two active X chromosomes (XaXa) is a hallmark of the ground  state of pluripotency specific to murine embryonic stem cells (ESCs). Human  ESCs invariably exhibit signs of X chromosome inactivation (XCI) and are  considered developmentally more advanced than their murine counterparts. We  describe the establishment of the first XaXa hESCs derived under physiological  oxygen concentrations.  Using these cell lines we demonstrate that (1)  differentiation of hESCs induces random XCI in a manner similar to murine  ESCs, (2) exposure to atmospheric oxygen is sufficient to induce irreversible XCI  and dramatic changes throughout the transcriptome, (3) the Xa exhibits heavy  methylation of the XIST promoter region, and (4) XCI is associated with  demethylation and transcriptional activation of XIST along with H3K27-me3  deposition across the Xi. These findings suggest that XaXa cells exist in the ICM  of the human blastocyst and that this state is preserved in vitro through culture  under physiological oxygen.

overall_design: Gene expression profiling was performed on human ES cell lines isolated and maintained under different oxygen concentrations

first: Christopher

last: Lengner

first: Jennifer

last: Erwin

first: Alexander

last: Gimelbrant

first: Matthew

last: Guenther

first: G

last: Welstead

first: Raaji

last: Alagappan

first: Garett

last: Frampton

first: Ping

last: Xu

first: Dong

last: Powers

middle: B

last: Barrett

first: Richard

last: Young

first: Jeannie

last: Lee

first: Rudolf

last: Jaenisch

first: Maisam

last: Mitalipova


title: Gene expression profiles following TP53 downregulation

status: Public on Mar 31 2010

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

country: Netherlands

institute: Vrije Universiteit Medical Center

summary: In three different ts-LT keratinocyte lines, TP53 was downregulated in four different ways. The vector constructs used for p53 abrogation were pWZL-p53DN-hygro (encoding the human p53 dominant-negative mutant Arg(175)His, pRS-p53shRNA-puro (containing a short hairpin RNA specific for p53), LZRS-E6-GFP (containing the HPV16-E6 oncogene) and the pRetrosuper miR-372. (containing the MicroRNA 372). Cells were transduced at 32°C as described above and checked for transduction efficiency by using an LZRS-GFP control. Only for miR-372 transduction efficiency showed to be below 90% and necessitated selection of positive cells by 1% blasticidin treatment for 5 days. Cells were harvested synchronously after two week culturing at 39°C.

overall_design: After RNA isolation, for each sample, two aliquots of 500ng of RNA were linearly amplified and fluorescently labeled with either Cy3-CTP or Cy5-CTP with the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Amstelveen, The Netherlands). Equal amounts (1µg) of Cy3-CTP and Cy5-CTP labeled samples were hybridized to Agilent 4x44K Whole Human Genome arrays (Agilent Technologies) according to the manufacturer. The sample set-up on the 4x44K array was chosen in such a way that all variables (cell lines and treatments) were on each array in dye swab and in duplicate on a different array. Equal amounts (1µg) of Cy3-CTP and Cy5-CTP labeled samples were hybridized to Agilent 44K Whole Human Genome arrays (Agilent Technologies, Part Number G4112A) according to manufacturer's instructions. Microarrays were scanned using an Agilent DNA Microarray Scanner, and scans were quantified using Agilent Feature Extraction software (version 8.5.1). Raw expression data generated by the Feature Extraction software was imported into R using the LIMMA package in Bioconductor (http://www.bioconductor.org). As overall background levels were very low, no background correction was performed. The intensity distributions within and between arrays were normalized using the quantile scaling algorithm in LIMMA. After normalization, the separate intensity channels were extracted from the ratio measurements and combined to the intensities of the reference. We assumed that overexpression of dominant-negative mutant p53, viral oncogene E6 and miR372 would have increased activity versus shRNA inactivation, and therefore analyzed all by expression profiling using the shRNA manipulated cells as reference.

first: Serge

last: Smeets

first: Koen

last: Bossers

first: Eva

last: van Veen

first: Paul

last: Eijk

first: Ruud

middle: H

last: Brakenhoff

first: Boudewijn

last: Braakhuis


title: Multiomics study to identify virulence factors of Rickettsia prowazekii revealed its adaptive mutation capabilities

status: Public on Mar 29 2010

city: Marseille

first: Yassina

last: Bechah

address: 27 bd J. Moulin

institute: CNRS

summary: We identified four virulence phenotypes of Rickettsia prowazekii (the deadly agent of epidemic typhus) that are associated with the upregulation of antiapoptotic genes (virulent strain) or the Interferon I pathway (avirulent). Transcriptional and proteomic analyses of R. prowazekii linked surface protein expression and methylation with virulence. By sequencing a virulent strain and using comparative genomics, we found hotspots of mutations in homopolymeric tracts of poly(A) and poly(T)  that lead to gene split and inactivation and explain the loss of virulence in the vaccine strain. These areas of instability explains adaptive mutations leading to virulence recovery in the vaccine strain.

overall_design: Several approaches are combined including the transcriptome of endothelial cells (the target cells of Rickettsia prowazekii) in order to identify factors associated with virulence of Rickettsia prowazekii, the agent of epidemic typhus

first: Khalid

last: El Karkouri

first: Oleg

last: Mediannikov

first: Quentin

last: Leroy

first: Nicolas

last: Pelletier

first: Cathrine

last: Robert

first: Claudine

last: Médigue

first: Jean-Louis

last: Mege

first: Didier

last: Raoult