title: Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algor

status: Public on Apr 24 2008

city: Baltimore

first: Minoru

last: Ko

state: MD

address: 251 Bayview Blvd, Suite 100, 10C

institute: NIH

summary:
We present a microarray experiment with the same tet-inducible system but with multiple time points within 24 hr to capture early responses to Oct4 suppression. These data were analyzed together with published genome-wide ChIP data. We found that most tentative target genes (TTG) of Oct4 in ES cells were activated by Oct4 and only a few genes were suppressed. The same method was then applied to find target genes of Sox2 and Nanog based mostly on previously published data. Because the interaction between Oct4, Sox2, and Nanog is supported by immunoprecipitation, functional analysis, and co-localization of binding sites, we explored the relationships between their target genes.
Keywords: time series design

overall_design: We used ZHBTc4 ES cells with a Tet-inducible Oct4 transgene. Cells were cultured  for 2 passages on gelatin-coated plates in order to remove feeder cells and then transferred to gelatin-coated 6-well plates at the density of 1-2x105 cells/well  and cultured in complete ES medium. Tetrocycline (TC) was added at 24 hr after cell plating, and then cells were harvested at 0hr (before adding TC), 3 hr, 6 hr, 12 hr, and 24 hr (2 replications each). RNA samples for later time points (24, 48, 72, 96, and 120 hr) were obtained from our earlier experiment with 3 replications. Two Nanog over-expressing clones were tested: (1) integrated transgene in MG1.19 parental cell line and (2) episomal transgene in EBRTcH3 parental line. EBRTcH3 and MG1.19 ESC lines were kind gifts of Dr. Hitoshi Niwa (RIKEN Center for Developmental Biology, Kobe, Japan). Both transgenic and parental cell lines were cultured  for 2 passages on gelatin-coated plates and then transferred to gelatin-coated 6-well plates at the density of 1-2x105 cells/well and cultured for 3 days in 3 different conditions: (1) complete ES medium (see above); (2) complete medium without LIF, and (3) complete medium with 1 uM RA. Cells were cultured at 37 0C and 5% CO2 condition and the culture medium was changed daily. Over-expression of Nanog was evaluated by PCR. Total RNAs were extracted using TrizolTM (Invitrogen, USA) and Phase lock gelTM columns (Eppendorf/Brinkman) according to the manufacturer???s protocol. Total RNAs were precipitated with isopropanol, washed with 70% ethanol, and dissolved in DEPC-treated H2O. 2.5 ug of total RNA samples were labeled with Cy3-CTP using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent, USA). A reference target (Cy5-CTP-labeled) was prepared from the Universal Mouse Reference (UMR) RNA (Stratagene, USA).

first: Alexei

last: Sharov

first: Shinji

last: Masui

first: Lioudmila

last: Sharova

first: Yulan

last: Piao

first: Ryo

last: Matoba

first: Hitoshi

last: Niwa


title: High oxygen prevents fetal lethality due to lack of catecholamines

status: Public on Jun 03 2008

city: Durham

first: Alisa

last: Ray

state: NC

address: DUMC Box 3209

department: Neurobiology

country: USA

institute: Duke University

summary:
The catecholamine norepinephrine is required for fetal survival, but its essential function is unknown.  When catecholamine-deficient [tyrosine hydroxylase (Th) null] mouse fetuses die at E13.5-E14.5, they resemble wild type fetuses exposed to hypoxia. They exhibit bradycardia (28% reduction in heart rate), thin ventricular myocardium (20% reduction in tissue), epicardial detachment, and death with vascular congestion, hemorrhage and edema. At E12.5, prior to the appearance of morphological deficits associated with Th deletion, catecholamine-deficient fetuses are preferentially killed by experimentally-induced hypoxia and have lower tissue pO2 than wild type siblings.  Catecholamine-deficient fetuses also induced HIF-1 target genes to a greater extent than wild type siblings, supporting the notion that null fetuses experience greater hypoxia or have an enhanced response to hypoxia. Hypoxia induces a 13-fold increase in plasma norepinephrine levels, which would be expected to increase heart rate, thereby, improving oxygen delivery in wild type mice. Surprisingly, increasing maternal oxygen (FiO2 33% or 63%) prevents the effects of catecholamine-deficiency, restoring heart rate, myocardial mass and survival of Th null fetuses.  We suggest that norepinephrine mediates fetal survival by maintaining oxygen homeostasis as vulnerability to constitutive hypoxia increases as fetal growth accelerates during normal development.
Keywords: Comparative with respect to genotype and oxygen conditions

overall_design:
The first aim was to compare gene expression of E12.5 mouse fetuses between wild type (Th+/+) versus tyrosine hydroxylase null (Th-/-) animals from normoxic dams. This resulted in 6 arrays.
The second aim compared wild type and tyrosine hydroxylase null E12.5 fetuses from dam exposed to hypoxia (8% oxygen) for 6 hours prior to sacrifice at E12.5 of gestation. This resulted in 6 arrays.
The third aim was to compare HIF-1 targets behind wt and null fetuses

first: Margie

last: Ream

first: Rashmi

last: Chandra

first: Mary

last: Peavey

first: Hyung-Gun

last: Kim

first: William

last: Wetsel

first: Howard

last: Rockman

first: Dona

last: Chikaraishi


status: Public on May 27 2008

city: Boston

first: Jonghwan

state: MA

address: 1 Blackfan circle Karp-7

institute: HMS/CHB

type: Genome binding/occupancy profiling by genome tiling array

summary:
Myc ChIP in HeLa cells and 2091 fibroblasts
Keywords: ChIP-chip

overall_design: HeLa cells, quiescent 2091 fibroblasts, and serum stimulated 2091 fibroblasts were used for Myc ChIP reactions.

first: Vishwanath

middle: R

last: Iyer


title: The tumor suppressor Semaphorin 3B triggers a pro-metastatic program mediated by IL-8 and the tumor microenvironment

status: Public on Apr 10 2008

city: Candiolo

first: Enzo

last: Medico

state: TO

address: Strada Prov. 142, km 3,95

department: Oncology

country: Italy

institute: Candiolo Cancer Institute, University of Torino

summary:
Semaphorins are a large family of evolutionary conserved morphogenetic signals originally identified as repelling cues in axonal guidance.  Intriguingly, Semaphorins have recently been implicated to play a role in cancer progression. In particular, Semaphorin 3B (SEMA3B) is considered a putative tumor suppressor, and yet we found that it is expressed at high levels in many invasive and metastatic human cancers. By investigating experimental tumor models, we confirmed that SEMA3B expression inhibited tumor growth, whereas metastatic dissemination was surprisingly increased. Interestingly, we found that SEMA3B induced the production of Interleukin-8 (IL-8) by tumor cells, by activating p38-MAPK pathway, in a Neuropilin-1 dependent manner. Further, by silencing the expression of endogenous SEMA3B in tumor cells, IL-8 transcription was impaired. The release of IL-8, in turn, induced tumor-associated macrophage recruitment and metastatic dissemination to the lung that could be rescued by using neutralizing antibodies against IL-8. In conclusion, we report here that SEMA3B plays unexpected functions in cancer progression, as it fosters a pro-metastatic environment, through elevated IL-8 secretion and recruitment of macrophages, coupled to suppression of tumor growth.
By performing micro-array analysis of gene expression on mRNA derived from MDA-MB-435 cells transduced with SEMA3B or control Empty Vector (EV), we found that one of the most regulated genes was Interleukin-8 (IL-8) (p< 0.001). Interestingly, IL-8 is known to be a chemo-attractant for leucocytes, endothelial and smooth muscle cells, further implicated in human cancer biology. We thus confirmed micro-array data by real-time quantitative PCR in tumor cell lines transduced with SEMA3B: i.e. A549 lung carcinoma, HeLa cervical carcinoma, HT29 colon carcinoma, and GTL16 gastric carcinoma. In addition, western blotting revealed that IL-8 secretion was strongly induced upon SEMA3B expression. Moreover, IL-8 protein levels were specifically up-regulated by treatment with doxycycline in A549 carcinoma cells expressing SEMA3B under control of a drug-inducible promoter.
Keywords: Cell type comparison

overall_design: A total of four samples were analyzed, representing biological duplicates of two conditions: cells transduced with empty vector or transduced with Sema3B expression vector

first: Luca

last: Tamagnone


title: Module map of stem cell genes guides creation of epithelial cancer stem cells

city: Stanford

first: David

last: Wong

state: CA

address: 269 Campus Drive

institute: Stanford University

summary:
Self-renewal is a hallmark of stem cells and cancer, but existence of a shared stemness program remains controversial. Here we construct a gene module map to systematically relate transcriptional programs in embryonic stem cells (ESCs), adult tissue stem cells, and human cancers. This map reveals two predominant gene modules that distinguish ESCs and adult tissue stem cells. The ESC-like transcriptional program is activated in diverse human epithelial cancers and strongly predicts metastasis and death. c-Myc, but not other oncogenes, is sufficient to reactivate the ESC-like program in normal and cancer cells. In primary human keratinocytes transformed by Ras and IkBa, c-Myc increases the fraction of tumor-initiating cells by 150-fold, enabling tumor formation and serial propagation with as few as 500 cells. c-Myc-enhanced tumor initiation is cell-autonomous and independent of genomic instability. Thus, activation of an ESC-like transcriptional program in differentiated adult cells may induce pathologic self-renewal characteristic of cancer stem cells.
A genetic modification design type is where an organism(s) has had genetic material removed, rearranged, mutagenized or added, such as knock out.
Keywords: genetic_modification_design, array CGH

overall_design: Computed

middle: J


title: Gene expression signature of cerebellum hypoplasia in a mouse model of Down syndrome (Part II).

status: Public on Mar 31 2008

city: Paris

first: Julien

last: Laffaire

address: 10 rue Vauquelin

country: France

institute: ESPCI - CNRS UMR 7637

summary:
We designed a large scale gene expression study in cerebellar external granular layer in Ts1Cje mice at P0 in order to measure the effects of trisomy 21 on in a enriched cell population (dissected layer) that is affected in Down syndrome in order to correlate gene expression changes to the phenotype observed.
Keywords: Down syndrome, Ts1Cje, EGL, hypoplasia

overall_design: We analyzed gene expression in the EGL of Ts1Cje and euploid mice at P0 using pangenomic Illumina mouse-6 v1.1 expression beadchips containing 46 632 probes representing approximately 19 000 mouse genes. 18 samples from individual cerebellar EGL were hybridized on 18 microarrays (6 by slide). On each slide, we hybridized 6 samples frome the same litter.

first: Isabelle

last: Rivals

first: Luce

last: Dauphinot

first: Tania

last: Vitalis

first: Jean

last: Rossier

first: Yann

last: Herault

first: Marie-Claude

last: Potier


title: Gene expression in colon cancer cell lines

status: Public on Mar 29 2008

city: SINGAPORE

first: qiang

last: yu

address: BIOPOLIS STREET

country: Singapore

institute: GIS

summary:
Genetic and epigenetic defects in Wnt/ß-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/ß-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly de-repressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/ß-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/ß-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/ß-catenin signaling in colorectal cancer.
Keywords: Colon cancer cell line

overall_design: Gene expression profile in HT29, DLD1 and SW480

first: Qiang

last: Yu


title: Colon cancer

summary:
Genetic and epigenetic defects in Wnt/?-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/ß-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly de-repressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/?-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/ß-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/ß-catenin signaling in colorectal cancer.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: aTEA 40 uM 12 hour treatment for MDA-MB-435 cells

status: Public on Mar 17 2008

city: Austin

first: Weiping

state: TX

address: 1 University Station

department: Microbiology

institute: The University of Texas at Austin

summary:
In an effort to identify novel signaling molecules modulated by α-TEA, microarray analyses were performed.  DNA array data obtained from human MDA-MB-435 breast cancer cells treated with 40 µM α-TEA for 12 h revealed over 400 genes that were consistently either up- or down-regulated. Thirty-four genes were selected based on their possible involvement in the known biological activities of α-TEA, and three: phorbol-12-myristate 13-acetate-induced protein (PMAPI) which codes for the protein NOXA, ABL2 which codes for the protein referred to as ARG (Abelson-related gene) and THBS1 which codes for thrombospondin 1, TSP-1 were chosen for further study.
Keywords: Anti-cancer drug (aTEA at 40 uM 12 hour) treatment for MDA-MB-435 cells

overall_design: The whole-genome cDNA microarrays were manufactured in the laboratory of Dr. Vishwanath Iyer (U.T. Austin). For the microarray experiments, MDA-MB-435 cells were cultured with 40 mM of a-TEA or ethanol control for 12 h. Messenger RNA was isolated using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA) then reverse transcribed by SuperScript II reverse transcriptase (Invitrogen) according to the manufacturers’ instructions. cDNA was purified using MinElute columns (Qiagen, Valencia, CA) and cDNA samples from vehicle treated controls were labeled with Cy3 and samples from a-TEA treated cells were labeled with Cy5. Array hybridizations were performed in humidity chambers at 65°C for 16 h. Slides were then washed, dried, and scanned using an Axon GenePix 4000 scanner (Axon Instruments, Union City, CA). Microarray images were quantified using GenePix 4.0 software (Axon). Data were uploaded to the Longhorn Array Database and filtered to pass minimum quality control thresholds before subsequent analysis. For a single comparison between two groups, a log2-transformed treatment/control signal ratio of  1 or –1 was chosen as the criterion for induction or repression, respectively. Data from 5 replica experiments were combined to eliminate variability and reduce misclassification.

first: Pei

last: Wang

first: Zhanzhi

last: Hu

first: Bob

middle: G

last: Sanders

first: Kimberly

last: Kline


title: Comparing gene expression in human colon tumor vs paired normal colon mucosa

summary:
24 colon normal and tumor pairs using Illumina BeadChip Human Ref8-v2.

Genetic and epigenetic defects in Wnt/ß-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/ß-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly de-repressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/ß-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/ß-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/ß-catenin signaling in colorectal cancer.

The clinical information for the colon tumor is not available.
Keywords: human colon tumor

overall_design: paired tumor vs. normal colon samples


title: HDAC Inhibitors Correct Frataxin Deficiency in a Friedreich Ataxia Mouse Model

status: Public on Mar 08 2008

city: Los Angeles

first: Giovanni

last: Coppola

address: 1524 Gonda, 695 Charles Young Drive South

department: Psychiatry and Neurology

institute: UCLA

summary:
Background: Friedreich ataxia, an autosomal recessive neurodegenerative and cardiac disease, is caused by abnormally low levels of frataxin, an essential mitochondrial protein. All Friedreich ataxia patients carry a GAA/TTC repeat expansion in the first intron of the frataxin gene, either in the homozygous state or in compound heterozygosity with other loss-of-function mutations. The GAA expansion inhibits frataxin expression through a heterochromatin-mediated repression mechanism. Histone modifications that are characteristic of silenced genes in heterochromatic regions occur at expanded alleles in cells from Friedreich ataxia patients, including increased trimethylation of histone H3 at lysine 9 and hypoacetylation of histones H3 and H4.
Methodology/Principal Findings: By chromatin immunoprecipitation, we detected the same heterochromatin marks in homozygous mice carrying a (GAA)230 repeat in the first intron of the mouse frataxin gene (KIKI mice). These animals have decreased frataxin levels and, by microarray analysis, show significant gene expression changes in several tissues. We treated KIKI mice with a novel histone deacetylase inhibitor, compound 106, which substantially increases frataxin mRNA levels in cells from Friedreich ataxia individuals. Treatment increased histone H3 and H4 acetylation in chromatin near the GAA repeat and restored wild-type frataxin levels in the nervous system and heart, as determined by quantitative RT-PCR and semiquantitative western blot analysis. No toxicity was observed. Furthermore, most of the differentially expressed genes in KIKI mice reverted towards wild-type levels.
Conclusions/Significance: Lack of acute toxicity, normalization of frataxin levels and of the transcription profile changes resulting from frataxin deficiency provide strong support to a possible efficacy of this or related compounds in reverting the pathological process in Friedreich ataxia, a so far incurable neurodegenerative disease.
Keywords: drug response

overall_design: Two mouse strains (KIKI and KO) were treated with compound 106 or control. Three tissues (Brain, Cerebellum, Heart) were examined in quadruplicate, for a total 48 arrays.

first: Myriam

last: Rai

first: Elisabetta

last: Soragni

first: Kai

last: Jenssen

first: Ryan

last: Burnett

last: Herman

first: Daniel

middle: H

last: Geschwind

first: Joel

middle: M

last: Gottesfeld

first: Massimo

last: Pandolfo


title: Early mouse embryo development

status: Public on Mar 25 2008

city: Toronto

first: Brian

middle: Joseph

last: Cox

state: Ontario

address: 1 King's College Circle, Rm 3360

department: Physiology

country: Canada

institute: University of Toronto

summary:
Expression profiling of mouse embryos at E2.5, E3.5, E4.25, E4.5, E5.5 E6.0 to identify genes regulated during development of the ICM (inner cell mass), TE (Trophectoderm) and PrE (Primative endoderm)
Keywords: development

overall_design: dupicate analysis of time points

first: Claire

last: Chazaud

first: Janet

last: Rossant


title: Identification of EMT related genes in mcf10 cell line

status: Public on Mar 24 2008

city: Bron

first: Hector

last: Hernandez-Vargas

state: Rhone

address: 36 bis Rue de la Batterie

institute: Genomics Consulting

summary:
In vitro studies on immortalized breast MCF10A cells were additionally performed to assess the phenotypic and genetic changes associated to mesenchymal transformation of breast cells and to their epithelial plasticity.
Keywords: breast cancer

overall_design: To further study the molecular events associated to the cell plasticity of MCF10A cells, we decided to characterize the genetic programs modulated during the phenotypic changes by cDNA microarrays analysis. Cells were grown to 10-20% (sparse) or 80-100% confluence (confluent), and the corresponding RNAs were extracted and hybridized onto cDNA microarrays.

first: Gema

last: Moreno-Bueno


status: Public on Feb 27 2008

city: Karlsruhe

first: Michael

middle: J.

last: Pankratz

department: Institute of Genetics

country: Germany

institute: Forschungszentrum Karlsruhe

summary:
Background
Insulin signaling pathway is conserved from worms to humans. In Drosophila, three insulin-like peptides (dilps) are expressed in the insulin producing cells (IPCs) in the brain. In order to identify target genes of insulin signaling,  the IPCs of female flies were destroyed during development by expressing apoptosis inducing factors reaper and head involution defective under control of the promoter region of dilp3. These insulin-deficient flies were analysed using high density microarrays.

Results
From the genes that appeared to be regulated in the absence of IPCs, the strongest difference was found for a gene involved in carbohydrate metabolism. Sequence analysis revealed that it is an alpha-glucosidase, which is able to break down disaccharides and/or glycogen.

Conclusions
We could identify an alpha-glucosidase, involved in carbohydrate metabolism, to be strongly affected in the context of decreased insulin signaling. The striking feature of this regulation was not only that it was the highest regulated, but it was the only gene regulated to this degree.

Nutrient Conditions and Fly handling
Experimental and control female flies were collected after hatching, then kept on fly maintenance food and separated from males after 11 days. After one day of recovery from carbon dioxide treatment, females were put in cages with PBS agar plates and supplied with yeast paste as only food source for 48 hours. Flies were shock frozen in liquid nitrogen and stored at - 80°C for further handling.
Keywords: IPC knockout, 14 day old female flies, 48h yeast feeding

overall_design:
Insulin producing cell deficient flies were compared to control flies. Two independent biological repeats were performed. From the two repeats, 5 chips were hybridised, including dye swap

Experimental and control female flies were collected after hatching, then kept on fly maintenance food and separated from males after 11 days. After one day of recovery from carbon dioxide treatment, females were put in cages with PBS agar plates and supplied with yeast paste as only food source for 48 hours.
The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link.
Keywords = insulin, aging, longevity, nutrient dependance
Lot batch = FP7

first: Susanne

last: Buch

first: Christoph

last: Melcher

first: Matthias

last: Bauer

first: Jörg

middle: D

last: Katzenberger


title: Head and neck squamous cell carcinoma cell lines (HNSCC) compared with normal keratinocytes

status: Public on Mar 11 2008

city: Bethesda

first: Bin

last: Yan

address: 10 Center Dr. Bldg 10, Rm 5D55

department: NIDCD

institute: National Institute of Health

summary:
Gene expression profiles of human HNSCC lines were compared with human normal keratinocytes by 24K cDNA microarray. The ten HNSCC analyzed are derived by the University of Michigan (UM-SCC), representing late stage SCC of different anatomic sites. Principle component analysis and hierarchical gene clustering classified ten cancer cell lines into two subsets, and associated each of the subsets with a distinctive p53 status.
Keywords: Gene expression array-based

overall_design: Basal gene expression profiles were compared between ten cancer cell lines and normal keratinocytes from four individuals.  RNAs were isolated from cancer cell lines and keratinocytes. After reverse transcription, these fluorescently-labeled cDNA were mixed with a labeled cDNA of reference RNA followed by hybridization on each array slide.  Each array hybridization was repeated 2-4 times.

first: Xinping

last: Yang

first: Tin-Lip

last: Lee


title: Gene Expression Study in WT and ATF4 KO embryonic neurons

status: Public on Mar 01 2008

first: Giovanni

last: Coppola

summary:
Oxidative stress is pathogenic in neurological diseases including stroke. The identity of oxidative stress-inducible transcription factors and their role(s) in propagating the death cascade are poorly understood. Microarray analysis of neurons undergoing oxidative stress showed significant induction of prodeath genes. These genes have been shown to be regulated by the bZip transcription factor, ATF4. ATF4 protein localized to the promoter of a putative death gene in neurons in vitro and in vivo. Germline deletion of ATF4 in neurons resulted in a reduction in oxidative stress-induced gene expression and resistance to oxidative death. ATF4 knockout mice experienced significantly smaller infarcts and improved behavioral recovery as compared to wild-type mice subjected to the same reductions in blood flow in a rodent model of stroke. ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative death by ATF4 deletion was associated with decreased consumption of  the antioxidant glutathione. Restoration of ATF4 protein in knockout neurons was sufficient to restore sensitivity to oxidative death and to reaccelerate loss of glutathione. Together, these findings establish ATF4 as a redox-regulated, pro-death transcriptional activator in the nervous system that propagates death responses to oxidative stress in vitro and to stroke in vivo.
Keywords: ATF4, oxidative stress, gene expression, neuroprotection, stroke

overall_design:
WT and ATF4 KO embryonic neurons were studied.
Untreated neurons (no=8 per genotype) and neurons challenged with oxidative stress with the glutamate homolog homocysteate (HCA, no=4 per genotype) were studied, for a total of 24 samples.

first: Philipp

middle: S

last: Lange

first: Juan

middle: C

last: Chavez

first: John

middle: T

last: Pinto

first: Chiao-Wang

last: Sun

first: Tim

last: Townes

first: Rajiv

last: Ratan


title: Inhibition of phosphatidylinositol 3-kinase signaling in proliferating human T98G cells

status: Public on Jan 24 2008

first: Geoffrey

last: Cooper

address: 5 Cummington St

department: Biology

institute: Boston University

summary:
Continuous PI 3-kinase signaling is required for cell proliferation and survival, but the gene expression program controlled by PI 3-kinase in proliferating cells has not been elucidated.  We used microarray analyses to characterize the changes in gene expression resulting from inhibition of PI 3-kinase in actively proliferating cells.  Following 2 and 4 hours of PI 3-kinase inhibition, the time around which apoptosis initiated, 32 genes were significantly up-regulated and 53 down-regulated.  After 8 hours of inhibition, the total number of expression changes increased to over 200.  The genes regulated by inhibition of PI 3-kinase in proliferating cells were distinct from genes induced by growth factor stimulation of quiescent cells and included key regulators of cell proliferation and programmed cell death.
Keywords: time course

overall_design: Transcriptional profiling time course of proliferating human T98G cells following LY294002 treatment. RNA was harvested at 2, 4 and 8 hr following LY294002 treatment.  Two-color microarrays with dye-swap; 3 biological replicates per condition, independently grown and harvested. One replicate per array.

first: Jolyon

last: Terragni

first: Julie

last: Graham

first: Kenneth

middle: W

last: Adams

middle: E

last: Schaffer


title: Evaluation of expression quantitative trait loci within two interacting blood pressure quantitative trait loci

status: Public on Mar 04 2008

city: Washington, DC

first: Norman

state: DC

address: 2300  I Street, N.W.

department: Pharmacology & Physiology

institute: The George Washington University Medical Center

summary:
Genetic dissection of the S rat genome has provided strong evidence for the presence of two interacting blood pressure (BP) quantitative trait loci (QTLs), termed QTL1 and QTL2, on rat chromosome 5. However, the identities of the underlying interacting genetic factors remain unknown. Further experiments targeted to identify the interacting genetic factors by the substitution mapping approach alone are difficult because of the interdependency of natural recombinations to occur at the two QTLs.  We hypothesized that the interacting genetic factors underlying these two QTLs may interact at the level of gene transcription and thereby represent expression QTLs (eQTLs).  To detect these interacting eQTLs, a custom QTL chip containing the annotated genes within QTL1 and QTL2 was developed and used to conduct a transcriptional profiling study of S and two congenic strains that retain either one or both the QTLs.  The results uncovered an interaction between two transcription factors, DMRTA2 and NFIA.  Further, the ‘biological  signature’ elicited by these two transcription factors was differential between the congenic strain that retained LEW alleles at both QTL1 and 2 compared to the congenic strain that retained LEW alleles at QTL1 alone.  A network of transcription factors potentially affecting BP could be traced, lending support to our hypothesis.
Keywords: rat, hypertension, genetics, polygenic trait, microarray, gene expression

overall_design:
Pairs of Cy5 and Cy3 labeled targets were co-hybridized onto either a custom long oligonucleotide microarray for the interrogation of 231 genes encompassed by QTL1 and QTL2, or a TIGR rat cDNA array consisting of 26,401 probe elements representing 20,465 unique non-QTL genes. A “flip-dye” or “balanced block” design was used as the experimental method of choice to account for potential dye-bias labeling effects. Six “balanced block” normalized files are submitted for the long oligonucleotide array interrogating the hearts from S versus S.LEW(5)x6x9 animals, fourteen “flip-dye” hybridizations are submitted for the cDNA array interrogating the hearts from S versus S.LEW(5)x6x9 animals, and twelve “flip-dye” hybridizations are submitted for the cDNA array interrogating the hearts from S versus S.LEW(5)x6x11 animals.

GPR and MEV files cannot be located.

first: Bina

last: Joe

first: Bryan

last: Frank


title: Effect of PACAP treatment on Bovine chromaffin cells

first: Babru

middle: Bahan

last: Samal

address: 9000 Rockville Pike

department: SMN

institute: NIMH/NIH

summary:
The effect of PACAP treatment on the bovine chromaffin cells in vitro were investigated using mouse Mm36kd3 arrays. Although chromaffin cells have indigenous PACAP, the experiment was designed to find out the effect of additional PACAP on additional activation or suppression of transcription.
Keywords: compound treatment design

overall_design: cy5 labelled RNA from PACAP treated bovine chromaffin cells were paired with cy3 labelled RNA from control chromaffin cells for hybridization. Four biological repeats were carried out.


title: T-2 Toxin treatment with 108 ppm for 2 h

status: Public on Jan 15 2008

city: Tsukuba

first: Emiko

last: Kitagawa

state: Ibaraki

address: Onogawa 16-1

country: Japan

institute: National Institute of Advanced Industrial Science and Technology

summary:
The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous
benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and
monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. In order to evaluate toxicity by newly synthesized or mixture chemicals, toxicity-induced gene expression alteration profiles by known chemicals should be collected. In our study, cells need to be exposed with same experimental cellular condition, semi lethal (IC50), respectively. In the case of T-2 toxin(CAS; 21259-20-1), the exposure dose was decided as 108ppm by growth curve with continuously diluted exposure. T-2 toxin has beenn shpwn to cause
variety of toxic effect including inhibition of DNA, RNA and protein synthesis.
Keywords: stress response

overall_design: Series contains triplicate hybridization results from independent biological samples respectively.

first: YoungKyung

last: Kim

first: Yumiko

last: Iwahashi

first: Hitoshi


title: Mapping Promoter CpG Methylation in mESCs

status: Public on Jan 28 2008

first: Yin

last: Shen

address: 695 Charles E. Young Dr.

type: Methylation profiling by genome tiling array

summary:
DNA methylation and histone modifications influence gene activities in a variety of mammalian cells.  We report here the large scale mapping of DNA methylation patterns at proximal promoter regions in mouse embryonic stem cells (mESCs).  A majority of methylated genes represent cell differentiation-associated genes that are repressed in mESCs.  In contrast, unmethylated genes include many housekeeping and pluripotency genes such as STAT3 and Oct4/Nanog/Sox2, which are essential for ESC self-renewal.  By comparing methylation patterns to the binding domains of Oct4/Nanog, Polycomb proteins, and histone H3 lysine (K) 4/27 trimethylation on gene promoters, we found that promoter DNA methylation is frequently the only epigenetic marker associated with a subset of silenced genes in mESCs that do not possess H3 K4/K27 trimethylation marks and are free from the binding of Polycomb proteins and the Oct4/Nanog transcription factors.  Gene expression profiling of demethylated mESCs showed that a majority of up-regulated genes, including a subset of X-linked genes and many development genes, represent a distinct population of genes that are neither targeted by Polycomb and Oct4/Nanog, nor carry H3 K4/K27 trimethylation marks.  Our data suggest that promoter methylation patterns in mESCs represent a unique epigenetic program that complements other regulatory mechanisms to allow the expression of pluripotency genes, but represses a subset of genes that will only be activated in specific cell lineages upon cell differentiation.
Keywords: DNA Methylation, methylated DNA immunoprecipitation

overall_design: We compared DNA methylation patterns in Wildtype (J1) and DNA methylation deficient (TKO) mouse ES cells.  We performed 7 replicates using different passages of mouse ES cells.  For gene expression analysis, we used the same cultures and performed 3 replicates.  The supplied table is the average log ratio for the 3 replicates and takes into account the dye swap of replicate 2.

first: Shaun

last: Fouse

first: Matteo

last: Pellegrini

first: Steve

last: Cole

first: Alexander

last: Meissner

first: Leander

last: Van Neste

first: Rudolph

last: Jaenisch

first: Guoping

last: Fan


title: Transcriptional Activity in Lymph Nodes During the Initiation and Progression of Autoimmune Encephalomyelitis

status: Public on Apr 03 2008

city: San Francisco

first: Sergio

last: Baranzini

address: 513 Parnassus Ave

department: Neurology

institute: University of California San Francisco

summary:
NOD mice (8- to 13-wk-old) were bred and maintained at the La Trobe University Central Animal House (Bundoora, Melbourne, Australia). All experiments were conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes (National Health and Medical Research Council 1997), after approval by the La Trobe University Animal Ethics committee (Melbourne, Australia).  A total of 124 female mice were divided into three groups. The immunized group comprised 91 mice injected s.c. into the lower flanks with 200 micrograms of MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco). An i.v. injection of 350 ng of Bordetella pertussis toxin was administered both immediately thereafter and 48 h later. The 26 mice in the control group were injected with adjuvant and pertussis toxin only. The seven animals used for the baseline group (time, day 0) were naive, not injected mice.  Except for the seven naive animals from the baseline group, nine animals (seven from the EAE and two from the control group) were sacrificed (at the same time of the day) at each of 13 time points after immunization, which are detailed as days 3, 5, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, and 19 postimmunization.  Lymph nodes from the remaining four animals of the immunized and from the two control groups were dissected, immersed in RNAlater (Ambion), and frozen at 20C.  Lymph nodes were removed from RNAlater and homogenized in TRIzol (Invitrogen Life Technologies) using an electric homogenizer. After resuspending the final RNA pellet in water, samples were repurified using the RNeasy kit (Qiagen). cDNA was synthesized from 15 micrograms of total RNA using Superscript II RT (Invitrogen Life Technologies) and a modified dNTP mix containing dUTP. Samples were hydrolyzed by adding 10 microliters of 0.1 N NaOH, neutralized with 25 microliters of 1 M HEPES, and precipitated with 3 M sodium acetate and ethanol. Resuspension in 0.05 M sodium bicarbonate was followed by 1 h incubation with either N-hydroxysuccinimide ester Cy3 or Cy5 fluorescent dyes (Amersham Biosciences). Probes were quenched by the addition of 4 M hydroxylamine and neutralized with 100 mM sodium acetate. Final probe cleanup was conducted using the QIA Quick PCR purification kit (Qiagen). We followed a common reference design in which each Cy3-labeled lymph node probe was combined with a Cy5-labeled probe derived from a pool of RNA. Hybridization onto glass slides containing 18,240 spotted 60- to 70-mer oligonucleotides, followed by washing and scanning was performed at the Gladstone microarray core facility at the University of California (San Francisco, CA)
Keywords: Time series

overall_design: NOD mice (8- to 13-wk-old) were injected s.c. into the lower flanks with 200 micrograms of MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco). An i.v. injection of 350 ng of Bordetella pertussis toxin was administered both immediately thereafter and 48 h later. The mice in the control group were injected with adjuvant and pertussis toxin only.  Disease and control mice were sacrificed at 13 identical timepoints and microarrays were processed for each timepoint.

first: Jorge

last: Oksenberg

first: Claude

last: Bernard


title: Deoxynivalenol treatment with 10,000 ppm for 2 h

summary:
The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous
benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. In order to evaluate toxicity by newly synthesized or mixture chemicals, toxicity-induced gene expression alteration profiles by known chemicals should be collected. A lethal effect of a certain kind of mycotoxin is weak to yeast. The amount of exposure to yeast used the maximum dissolution density for afratoxin DON (Deoxynivalenol, CAS; 51481-10-8) (10,000 ppm, IC70）. Because the data that was able to be trusted was not obtained, it is not possible to have analyzed toxicity.
Keywords: stress response

overall_design: Series contains 4 hybridization results from independent biological samples respectively.


title: Analysis of effect of sirt1 deficiency on global gene expression in testis

status: Public on Feb 22 2008

city: Honolulu

first: rich

last: allsopp

state: HI

address: 651 Ilalo St

department: anatomy & reprod biol

institute: university of hawaii

summary:
Testis were obtained from 2 sibling pairs of of young male mice, 1 knock-out and 1 heterozygous for the Sirt1 allele, and used to compare global gene expression using spotted oligo microarrays (the MEEBO arrays from the Stanford microarray core- MOE series). Eighty genes were up or down regulated, where the criteria for differential expression was a greater than 2 fold change in expression level on at least 5 of the 6 sample arrays. The list of differentially expressed genes was enriched for genes involved in spermatogenesis.
Keywords: spotted oligonucleotide microarray, mouse

overall_design: 2 pairs of Sirt1 knock-out and heterozygous testis were analyzed. The global gene expression was assessed intriplicate for each pair, including 1 dye swap.

first: Richard

last: Allsopp

first: Matthew

last: Coussens

first: Greg

last: Maresh


title: Aflatoxin treatment with 10,000 ppm for 2 h

first: Emiko

last: Kitagawa

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

overall_design: Refer to individual Series


title: Transcriptional profiling of Control vs. Pinealectomised Syrian hamster testes

status: Public on Jan 08 2008

city: Cambridge

first: Akhilesh

middle: Basi

last: Reddy

address: Hills Road

department: Institute of Metabolic Science

country: United Kingdom

institute: University of Cambridge

summary:
Transcriptional profiling of male Syrian hamster testes, comparing control animals with those that have had a pinealectomy.
Keywords: transcriptional profiling

overall_design: Two-condition experiment, Control vs pinealectomised animals


title: Aflatoxin B1 treatment with 10,000 ppm for 2 h

summary:
The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous
benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. In order to evaluate toxicity by newly synthesized or mixture chemicals, toxicity-induced gene expression alteration profiles by known chemicals should be collected. A lethal effect of a certain kind of mycotoxin is weak to yeast. The amount of exposure to yeast used the maximum dissolution density for afratoxin B1 (CAS; 1162-65-8) (10000 ppm, IC80）. Because the data that was able to be trusted was not obtained, it is not possible to have analyzed toxicity.
Keywords: stress response

first: YoungKyung

last: Kim

first: Yumiko

last: Iwahashi

first: Hitoshi


title: Human podocyte differentiation

status: Public on Jan 05 2008

city: LYON

first: Jiri

last: ZAVADIL

state: Other

address: 150 COURS ALBERT THOMAS

institute: IARC WHO

summary:
Microarray analysis of the molecular phenotype of the glomerular podocyte during temperature shift-induced differentiation
Keywords = glomerular podocyte
Keywords = differentiation
Keywords: time-course


title: Comparison of gene expression profile of human trabecular meshwork cells induced by triamcinolone with dexamethasone

status: Public on May 01 2008

city: Boston

first: Bao Jian

address: MEEI, 243 Charles Street

department: Ophthalmology

institute: Harvard Medical School

summary:
PURPOSE. Triamcinolone acetonide (TA) and dexamethasone (DEX) are corticosteroids commonly used for ocular inflammation but both can cause ocular hypertension. We investigated the differential gene expression profile of human trabecular meshwork (TM) cells in response to treatment by TA compared to that by DEX.
METHODS. Total RNA was extracted from cultured human TM cells treated with TA or DEX and used for microarray gene expression analysis. The microarray experiments were repeated three times. Differentially expressed genes were identified by an empirical Bayes approach and confirmed by real-time quantitative PCR.
RESULTS. 0.1 mg/ml TA treatment resulted in 15 genes up-regulated and 12 genes down-regulated while 1mg/ml TA treatment resulted in 36 genes up-regulated and 21 genes down-regulated. These genes were mainly associated with acute-phase response, cell adhesion, cell cycle and growth, growth factor, ion binding, metabolism, proteolysis and transcription factor. Two genes, MYOC and GAS1, were up-regulated and 3 genes, SENP1, ZNF343 and SOX30, were down-regulated by both TA and DEX treatment. Eight differentially expressed genes were located in known primary open angle glaucoma (POAG) loci, including MYOC, SOAT1, CYP27A1, SPOCK, SEMA6A, EGR1, GAS1 and ATP10A.
CONCLUSIONS. Differential gene expression profiles of human TM cells treated by TA and DEX, and a dosage effect by TA, were revealed by microarray technology. TA and DEX treatment shared several differentially expressed genes, suggesting a common mechanism to cause ocular hypertension. Some differentially expressed genes located in the known POAG loci are potential candidates for glaucoma genes.
Keywords: Differential gene expression profile

overall_design: A total of 9 samples were used for this study. There were 3 biological replicates for each of 3 treatments.

first: Bao

first: Dan

middle: Y

last: Wang

first: Clement

last: Tham

first: Shun

last: Lam

first: Chi

middle: P

last: Pang


title: PACAP dependency of genes regulated in adrenal gland

first: Babru

last: Samal

summary:
We investigated the effects of PACAP knock out on the transcriptosome profile of adrenal gland. A 35,546-clone mouse cDNA microarray was used to investigate transcriptional changes
Keywords: transcript identification design

overall_design: In the hybridization method employed, equal amounts (2-5 ug/15.5 ul) of total RNA obtained from adrenal gland of wild type and PACAP KO mice groups were separately labeled with two different fluorescent dyes (Cy3 or Cy5) and applied on the same NIMH Mouse 36K cDNA microarray chip containing 13217 distinct Entrez gene IDs (formerly known as LocusLink) and 15888 distinct unigene IDs, the remainders are incompletely annotated IMAGE clones. Probe preparation and hybridization were performed as follows. The total RNA (15-50 ug in 15.5 ul) was incubated with amine-modified random primer (2 ug/ul, 2 ul) and RNase inhibitor (5 units/ul, 1 ul) at 70C for 10 min. Primer-RNA solution was then incubated at 42C for 2 hr with the reverse transcriptase mix containing 5X first-strand buffer, 50X aa-dUTP/dNTPs (25 mM dATP, 25 mM dGTP, 25 mM dCTP, 15 mM dTTP and 10 mM aminoallyl dUTP), 0.1 M DDT, and Superscript II reverse transcriptase. The cDNA were labeled with NHS-ester Cy3 or Cy5 dye in the presence of 1 M sodium bicarbonate. Array slides placed in a hybridization chamber (Corning, Corning, NY) were incubated at 42C for 16-24 hr, and successively washed with 0.5X SSC, 0.01% SDS, and 0.06X SSC at room temperature for 10 min each. Arrays were scanned with a GenePix 4000A scanner (Axon, Foster City, CA), and the resulting images were analyzed using IPlab (Fairfax, VA) and a FileMaker Pro 5 (Santa Clara, CA) relational database designed by Zedong Chen, NHGRI. Following background subtraction and normalization, a calculated ratio of Cy3 to Cy5 signal intensities was used to define the relative increase or decrease of a particular transcript. Ratios were calculated only from those spots with a combined ratio quality above 0.3.


title: Across array comparative genomic hybridization

status: Public on Dec 31 2007

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

country: Netherlands

institute: Vrije Universiteit Medical Center

summary:
Aim of this study is to test if a reference channel of a different array can be used to make array CGH more sensitive, cost effective and less labor intensive.  The BT474 breast cancer cell line was compared to a mix of normal reference DNAs hybridized on different arrays and days and DNA copy number profiles were evaluated. Quality was assessed, using regular dual channel array CGH as a standard, using four quality measures; i.e. the MAD (median absolute deviation) value of chromosome 2, an amplitude of the ErbB2 gene amplification, a deletion on chromosome 9 and a deflection on chromosome 8. In addition, the use of a reference channel of a different array was tested for genomic DNA derived from formalin-fixed paraffin-embedded tumor tissue samples. This so called across array CGH (aaCGH) analysis yielded slightly higher quality chromosomal copy number profiles when using the same dye compared to analysis using two different dyes, both when exchanging fluorescent signals of hybridizations performed on different arrays and on different days. This approach avoids redundant hybridizations of the same reference material in every experiment and allows hybridization of two test samples on one array. AaCGH analysis substantially reduces cost of consumables and labor while yielding at least equivalent quality as the dual channel procedure.
Keywords: array CGH data, across array CGH analysis

overall_design: Genomic DNA from the cell line BT474, two colorectal cancer (CRC) tissue samples (FFPE) and one gastric cancer (GC) tissue sample (FFPE) was isolated. DNA isolated from blood obtained from eighteen healthy males was mixed for the use as a normal reference. Hybridizations were performed on 4x44K arrays (agilent technologies), on different days. Hybridizations of BT474 versus normal reference were performed in triplicate, and also the dyeswap was performed in triplicate. In total, 24 single channel array CGH data was used to calculate log2 tumor to normal ratios

first: Tineke

last: Buffart

first: Thijs

last: Krugers

first: Marianne

last: Tijssen

first: Sjoerd

last: Vosse

first: Alan

last: Mršić

first: Gerrit

middle: A

last: Meijer

first: Bauke

last: Ylstra


title: Aflatoxin G1 treatment with 10,000 ppm for 2 h

first: Emiko

last: Kitagawa

summary:
The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous
benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. In order to evaluate toxicity by newly synthesized or mixture chemicals, toxicity-induced gene expression alteration profiles by known chemicals should be collected. A lethal effect of a certain kind of mycotoxin is weak to yeast. The amount of exposure to yeast used the maximum dissolution density for afratoxin G1 (CAS; 1165-39-5) （10,000 ppm, IC70）. Because the data that was able to be trusted was not obtained, it is not possible to have analyzed toxicity.
Keywords: stress response

first: YoungKyung

first: Yumiko

first: Hitoshi


title: Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells

status: Public on Mar 31 2008

city: Seattle

first: Vitalina

last: Komashko

state: WA

address: 1100 Fairview Ave N

institute: Sage Bionetworks

type: Expression profiling by genome tiling array

summary:
We compared 12 different cell populations, including embryonic stem cells before and during differentiation into embryoid bodies as well as various types of normal and tumor cells to determine if pluripotent vs. differentiated cell types use different mechanisms to establish their transcriptome. We first identified genes that were not expressed in the 12 different cell populations and then determined which of them were regulated by histone methylation, DNA methylation, at the step of productive elongation, or by the inability to establish a pre-initiation complex. For these experiments, we performed chromatin immunoprecipitation using antibodies to H3me3K27, H3me3K9, 5-methyl cytosine, and POLR2A. We found that a) the percentage of low expressed genes bound by POLR2A, H3me3K27, H3me3K9, or 5-methyl-cytosine is similar in all 12 cell types, regardless of differentiation or neoplastic state; b) a gene is generally repressed by only one mechanism; and c) distinct classes of genes are repressed by certain mechanisms. We further characterized two transitioning cell populations, 3T3 cells progressing from G0/G1 into S phase and mES cells differentiating into embryoid bodies. We found that the transient regulation through the cell cycle was achieved predominantly by changes in the recruitment of the general transcriptional machinery or by post-POLR2A recruitment mechanisms. In contrast, changes in chromatin silencing were critical for the permanent changes in gene expression in cells undergoing differentiation.
Keywords: ChIP-chip, RNA expression

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
48 ChIP-chip arrays; 12 expression arrays (6 of them in duplicates).
47 samples are included in this series, the rest can be found in supplementary info to the following papers: O'Geen 2007, Acevedo 2008, Acevedo 2007, Rinn 2007

first: Luis

last: Acevedo

first: Sharon

middle: L

last: Squazzo

first: Sushma

last: Iyengar

first: Alina

last: Rabinovich

first: Henriette

last: O'Geen

first: Roland

last: Green

first: Peggy

middle: J

last: Farnham


title: Effects of DKK1 on gene expression on keratinocytes

first: Vincent

last: Hearing

address: Bldg 37  Rm 2132

department: National Cancer Institute

institute: National Institutes of Health

summary:
Dickkopf 1 (DKK1), which is expressed at high mRNA levels by fibroblasts in the dermis of human skin on the palms and soles, inhibits the function and proliferation of melanocytes in the epidermis of those areas via the suppression of beta-catenin and microphthalmia-associated transcriptor factor (MITF). In this study, we report that treatment of keratinocytes with DKK1 increases their proliferation and decreases their uptake of melanin, and that treatment of reconstructed skin with DKK1 induces a thicker and less pigmented epidermis. We investigated the effects of DKK1 on keratinocytes using DNA microarray technology.
Keywords: compound treatment design

overall_design: Paired cDNA samples, labeled by cyanine 3- and cyanine 5-dUTP incorporation (Qiagen, Valencia, CA) during reverse transcription (Qiagen), were hybridized simultaneously with one oligo-DNA chip (HS-Operon V2vB2.2p13) as per NCI in-house protocol. Two fluorescent intensities of the oligo-DNA chip were scanned using a microarray scanner (GenePix 4000A, Axon Instruments, Inc., Molecular Device Corp., Sunnyvale, CA).

first: Yuji

last: Yamaguchi


title: Transcriptional targets of FOXP2 in human brain and lung

first: Daniel

middle: H

last: Geschwind

address: 695 Charles E Young Drive

institute: UCLA

summary:
Mutations in FOXP2, a member of the forkhead family of transcription factors, are the only known cause of developmental speech and language disorders in humans. To date, there are no known targets of human FOXP2 in the nervous system.  The identification of FOXP2 targets in the developing human brain therefore provides a unique tool with which to explore the development of human language and speech. Here we define FOXP2 targets in human basal ganglia (BG) and inferior frontal cortex (IFC) utilizing chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) and validate the functional regulation of targets in vitro.  ChIP-chip identified 285 FOXP2 targets in fetal human brain; significant overlap of targets in BG and IFC, indicate a core set of 34 transcriptional targets of FOXP2. We identified targets specific to the IFC or BG, not observed in lung, suggesting important regional and tissue differences in FOXP2 activity. Many target genes are known to play critical roles in specific aspects of CNS patterning or development, such as neurite outgrowth, as well as plasticity. Subsets of the FOXP2 transcriptional targets are either under positive selection in humans, or differentially expressed between human and chimpanzee brain. This is the first ChIP-chip study using human brain tissue, making the FOXP2 target genes identified in these studies important to understanding the pathways regulating speech and language in the developing human brain. These data provide the first insight into the functional network of genes directly regulated by FOXP2 in human brain and by evolutionary comparisons, highlight genes likely to be involved in the development of human higher order cognitive processes.
Keywords: ChIP-chip

overall_design: Three independent brain samples were used for both the basal ganglia and inferior frontal cortex experiments. Two different pieces from the same lung tissue were used.

first: Elizabeth

last: Spiteri

first: Genevieve

last: Konopka

first: Giovanni

last: Coppola

first: Jamee

last: Bomar

first: Sonja

last: Vernes

first: Jing

last: Ou

first: Simon

last: Fisher

first: Bing

last: Ren


title: BJAB Cell Lines Unmodified, Transduced with ith lentiviral vector pNL-SIN-CMV-AcGFP or pNL-SIN-CMV-AcGFP/miR-K12-11

status: Public on Dec 19 2007

city: Durham

first: Bryan

middle: R.

last: Cullen

address: CARL Bldg 427, Research Drive

institute: Duke University Medical Center

summary:
All metazoan eukaryotes express microRNAs (miRNAs), ~22 nt long regulatory RNAs that can repress the expression of mRNAs bearing complementary sequences. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis. While specific viral miRNAs have been shown to autoregulate viral mRNAs or downregulate cellular mRNAs via novel target sites, the function of the majority of viral miRNAs remains unknown. Here, we report that the miR-K12-11 miRNA encoded by Kaposi’s Sarcoma Associated Herpesvirus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA “seed” region. Using a range of assays, we demonstrate that expression of physiological levels of miRK12- 11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12-11 functions as an ortholog of cellular miR-155 and has likely evolved to exploit a pre-existing gene regulatory pathway in B-cells. Moreover, the known etiological role of miR-155 in B-cell transformation suggests the possibility that miR-K12-11 may contribute to the induction of KSHV positive B-cell tumors in infected patients.
Keywords: miRNA stable expression comparisons

overall_design:
BJAB cells were infected and sorted 48 hours after infection.
12 to 16 days after transduction, gene expression analysis of 10 independent BJAB cell pools, expressing AcGFP only or AcGPF and miR-K12-11, was performed using Human Operon v3.0.2 arrays. Each ample was run against Universal Human Reference RNA, Stratagene.
# of Arrays:
BJAB = 3
AcGFP only = 10
AcGFP miR-K12-11 = 11

first: Eva

last: Gottwein

first: Neelanjan

last: Mukherjee

first: Corina

last: Frenzel

first: Christoph

last: Sachsec

first: William

last: Majoros

first: Jen-Tsan

last: Chi

first: Juergen

last: Soutschek

first: Uwe

last: Ohler

middle: R


title: Messenger RNA profiling of bovine endometrium during the estrous cycle using a custom-made cDNA array

city: Lindau

first: Stefan

middle: Michael

last: Bauersachs

state: Zurich

address: Eschikon 27 EHB 23.1

department: Department for Farm Animals

country: Switzerland

institute: University of Zurich

summary:
The endometrium provides optimal conditions for the transport of sperm to the oviduct, to the site of fertilization, and later on for the reception of the embryo. To study these changes on the level of gene expression, a messenger RNA expression profiling of endometrium tissue samples collected from 19 cyclic heifers at five stages of the estrous cycle (days 0, 3.5, 12, 18, 20) was performed. RNA was extracted from these tissue samples and analyzed with a custom-made bovine oviduct and endometrium (BOE) cDNA array. The cDNAs present on the array were derived from several previously conducted differential gene expression studies of bovine endometrium between different stages of the estrous cycle, during early pregnancy, and from studies of bovine oviduct epithelial cells. In all of these studies cDNAs of differentially expressed genes were identified using a combination of subtracted cDNA libraries and cDNA array hybridization. 1,440 cDNA fragments are located on the array. Twenty radioactively labeled cDNA samples (n=4 for each cycle stage) were hybridized with BOE arrays. Raw data were normalized using the BioConductor package vsn.
Keywords: time course of gene expression in bovine endometrium during estrous cycle

overall_design: Nineteen German Fleckvieh (Simmental) heifers were slaughtered at four different days of the estrous cycle, four animals at day 0, four at day 3.5, three at day 12 and eight animals at day 18. Four of the day 18 animals showed high and the other four low serum progesterone (P4) levels, respectively. This resulted in five groups of endometrial tissue samples corresponding to five stages of the estrous cycle.

first: Katrin

middle: G

last: Mitko

first: Susanne

last: Ulbrich

first: Helmut

last: Blum

first: Eckhard

last: Wolf


title: Dependency of Gene Expression in Saccharomyces cerevisiae on extracellular Glucose Concentration

status: Public on Dec 02 2007

city: Bremen

first: Christine

last: Klockow

address: Celsiusstr. 1

institute: Max Planck Institute for Marine Microbiology

summary:
The goal of this study is to analyse how the gene expression in Saccharomyces cerevisiae changes in dependency on the glucose concentration. Therefore, fed batch cultivations were carried out, during which the glucose concentration was maintained stable for several hours. Samples were taken at different times during the cultivations, the RNA was isolated and hybridised on whole genome yeast microarrays. Results from cultivations with the glucose concentrations 50, 70, 100 and 500 mg/L are presented. In addition, one sample from a starvation period (0 mg/L glucose) was analysed.
Keywords: Dependency on glucose concentration

overall_design: Results from seven microarray are summarised in this study. The samples originate from four different cultivations with the glucose set points mentioned above. Supplementary, one sample from a starvation period is shown and during the cultivations with glucose set points 70 mg/L and 500 mg/L, samples drawn at different cultivation times are presented.

first: Frank

last: Stahl

first: Bernd

last: Hitzmann


title: Gene Expression Profile in Developing Mouse Brain Following Chronic Hypoxia Treatment

status: Public on Dec 04 2007

city: La Jolla

first: Dan

last: Zhou

address: CMG 103, 9500 Gilman Drive, MC0735

department: Pediatrics

institute: University of California, San Diego, School of Medicine

summary:
In this study, we determined the effect of chronic consistent hypoxia (CCH) and chronic intermittent hypoxia (CIH) on global gene expression in cortical and hippocampal region of developing mouse brain using long-oligo beadschip arrays.
Keywords: Gene expression profile

overall_design:
Control-Cortex: Cortical samples from 16 days old mice were analysed on whole-genome expression chips to reveal expression profile at this developmental stage.  Used as control for Chronic Hypoxia Treated cortical samples.

CCH-Cortex: Cortical samples from 2-week CCH treated mice were analysed on whole-genome expression chips to reveal the changes in gene expression profile following 2-week CCH treatment.

CIH-Cortex: Cortical samples from 2-week CCH treated mice were analysed on whole-genome expression chips to reveal the changes in gene expression profile following 2-week CIH treatment.

Control-Hippocampus: Hippocampal samples from 16 days old mice were analysed on whole-genome expression chips to reveal expression profile at this developmental stage.  Used as control for Chronic Hypoxia Treated hippocampal samples.

CCH-Hippocampus: Hippocampal samples from 2-week CCH treated mice were analysed on whole-genome expression chips to reveal the changes in gene expression following 2-week CCH treatment.

CIH-Hippocampus: Hippocampal samples from 2-week CCH treated mice were analysed on whole-genome expression chips to reveal the changes in gene expression following 2-week CIH treatment.

first: Jiyi

last: Wang

first: Gabriel

last: Haddad


title: Confluent versus Sub-confluent HUVEC

status: Public on Dec 01 2007

first: Oscar

middle: Leonard

last: Volger

state: Noor Holland

address: Meibergdreef 15

department: Cardiology

institute: Academic Medical Center

summary:
HUVEC (N=3 isolates) were separately grown to sub-confluency, and to confluency. In order to study the effects of growth and contact inhibition on the transcriptome, the microarray gene expression profiles of these sub-confluent and confluent HUVEC were compared.
Keywords: HUVEC, confluency, contact inhibition

overall_design: N=3 biological replicates, separately grown to sub-confluency, and concluency, respectively. Total RNA molecules were then isolated, and linearly amplified (1 round) according to the Eberwine method. After labeling the amplified cRNA molecules of these 6 samples (N=3 sub-confluent, N=3 confluent) with Cy5 Dyes, these were separately hybridized against Cy3-labeled common reference RNA molecules on glass-based 19K oligonucleotide microarray chips.

first: Ruud

middle: D

last: Fontijn

first: Joost

middle: O

last: Fledderus

first: Anton

last: Horrevoets


title: HU treated cells vs wildtype untreated cells (asynchronized cells)

status: Public on Dec 20 2007

city: Singapore

first: Zhaoqing

last: Chu

address: 60, Biopolis Streat

institute: Genome Institute of Singapore

summary:
DNA replication checkpiont kinase rad3 (human ATM/ATR-like kinase) and cds1 (human CHK2-like kinase) are known to restore stalled replication forks and prevent subsequent cell cycle progression during a replication block imposed by HU-treatment in fission yeast. In order to identify cell cycle-specific exression which are modulated by replication checkpoint kinase rad3 and cds1, microarray approach was used to profile asynchronous and synchronous cells of wildtype and mutants treated and untreated with HU
Keywords: HU treated cells vs wildtype untreated cells

overall_design: We analyzed 60 arrays for asynchronized cells of wildtype and rad3,cds1,chk1 mutants treated with 8 mM HU for 0,10,30,60 and 120 min,three biological repeats for each time point, 10 arrays for asynchronized cells of rad3sep1 double mutant treated with 8 mM HU for 0,10,30,60 and 120 mins,two biological repeat for each time point.

first: Juntao

last: Li

first: Krishna

middle: M

last: Karuturi

first: Jianhua

last: Liu


title: HU treated cells vs wildtype untreated cells (elutriation centrifugation synchronized cells)

overall_design: We analyzed 88 arrays for elutriation centrifugation synchronized cells of wildtype, rad3,cds1,and chk1 mutants release to the early G2 phase at 0,40,80,120,160 and 200 mins untreated (HU-) and treated (HU+) with 8 mM HU, two technique repeat for each time points


title: Short-term high fat-feeding results in morphological and metabolic adaptations in the skeletal muscle of C57BL/6J mice

status: Public on Nov 30 2007

city: Maastricht

first: Janneke

last: de Wilde

address: Universiteitssingel 50

department: Human Biology

institute: Maastricht University

summary:
The prevalence of the metabolic syndrome (MS) is rapidly increasing all over the world. Consequently, there is an urgent need for more effective intervention strategies. Both animal and human studies indicate that lipid oversupply to skeletal muscle can result in insulin resistance which is one of the charecteristices of the MS.
C57BL/6J mice were fed a low fat (10 kcal%) palm oil diet or a high fat (45 kcal%; HF) palm oil diet for 3 or 28 days. By combining transcriptomics with protein and lipid analyses we aimed to better understand the molecular events underlying the early onset of the MS.
Short-term HF-feeding led to altered expression levels of genes involved in a variety of biological processes including morphogenesis, energy metabolism, lipogenesis and immune function.
Protein analysis showed increased levels of the myosin heavy chain, slow fiber type protein and the complexes II, III, IV and V of the oxidative phosphorylation. Furthermore, we observed that the main mitochondrial membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine, contained more saturated fatty acids.
Altogether, these results point to a morphological as well as a metabolic adaptation by promoting a more oxidative fiber type. We hypothesize that after this early adaptation, a continued transcriptional down-regulation of genes involved in oxidative phosphorylation will result in decreased oxidative capacity at a later stage.
Together with increased saturation of phospholipids of the mitochondrial membrane this can result in decreased mitochondrial function which is a hallmark observed in insulin resistance and type 2 diabetes.
Keywords: diet intervention and time course

overall_design:
In the present study we investigated the short versus the long term effects of a high fat diet on the mouse muscle transcriptome by performing a genome-wide analysis of high fat diet-responsive genes.
C57BL/6J mice were fed a low fat (LF) diet (10 kcal% palm oil) or a high fat (HF) diet (45 kcal% palm oil) for 3 or 28 days. Total RNA samples (n = 6 per diet per time point) were individually hybridised tot the Affymatrix mouse genome 430 2.0 array.

first: Ronny

last: Mohren

first: Sjoerd

last: van den Berg

first: Mark

last: Boekschoten

first: Ko

last: Willems - Van Dijk

first: Philip

last: de Groot

first: Michael

last: Müller

first: Edwin

last: Mariman

first: Egbert

last: Smit


title: Cell Cycle-specific Expressions Are Modulated by Replication Checkpoint Kinases Rad3 and Cds1 in Fission Yeast

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


title: Gene Expression of Human Lymphocytes Used For ACT of Metastatic Melanoma

status: Public on Dec 15 2007

first: Keith

last: Kerstann

department: Surgery Branch

summary:
Recent reports have detailed the efficacy of the adoptive cell transfer (ACT) of autologous anti-tumor lymphocytes for the treatment of patients with melanoma refractory to standard therapy. Here we report the microarray analysis of tumor infiltrating lymphocytes (TIL) generated from twenty-eight melanoma patients enrolled in a treatment protocol that combines non-myeloablative lymphodepletion followed by adoptive cell transfer. The patients were divided into two groups Ÿ twelve who experienced an objective response according to RECIST criteria and sixteen who did not. Using several methods of data analysis, no consistent overt changes in gene expression, pathways or classical markers of lymphocyte development were detected between TIL administered to responding versus non-responding patients. Only one non Y chromosome gene, phosphoserine phophatase-like (PSPHL), was found to be over expressed primarily in lymphocytes that did not result in tumor regression and seemed to negatively correlate with in vivo survival of transferred TIL. As persistence of lymphocyte clonotypes in vivo was correlated with patient response, cyro-preserved treatment TIL cultures were bead-separated into in vivo persisting and non-persisting fractions. Subsequent microarray analysis of these persisting and non-persisting clonotypes also did not reveal any consistent differences in gene expression. Finally, to determine if the lymphocytes used in our adoptive cell transfer studies had similar gene expression profiles, the twenty-eight TIL samples were compared to 10 samples generated through limiting dilution cloning from an early ACT trial. This analysis revealed differences in the gene expression of lymphocytes that have been used in these two different ACT trials. However, the inability to find differential gene expression in tumor infiltrating lymphocytes that correlate to patient response or persistence suggests that differences affecting these clinical events lie in host or tumor factors rather than transferred cells.
Keywords: cell type comparison design

overall_design: There were 28 patient samples with 12 responding and 16 non-responding patients. There were 6 persisting and 6 non-persisting clonotypes. There were 10 cloned lymphocyte samples. All 50 samples were arrayed using Universal Reference as a control in a two-color hybridization.


title: Expression analysis of gene differentially expressed in the developping ovary

status: Public on Nov 21 2007

city: Beaverton

first: Valerie

last: Matagne

state: OR

address: 505 nw 185th Ave

department: Neuroscience

institute: OHSU

summary:
In recent years, several factors required for follicle assembly and/or early growth of newly formed primordial follicles have been characterized, but it is likely that additional factors remain to be identified. We have used  cDNA arrays to compare gene expression in the neonatal mouse ovary 48 h after birth (when primordial follicles are being assembled), and at 96 h (when early follicular growth is taking place), to that of ovaries collected less than 24 h after birth (when follicles have not yet been formed).  Segregating genes according to their pattern of expression revealed the presence of one cluster of 24 genes whose expression consistently increased at 48 and 96 h. The top increaser in this cluster encodes a ~1.5 Kb mRNA containing an open reading frame of 1,401 bp that encodes a protein of 466 amino acids. The predicted 52.3 kDa protein, which we have termed Eop1 (for Early ovarian protein-1) is a novel member of the F-box only (Fbxo) protein family. Eop1 has a cytoplasmic localization that includes the endoplasmic reticulum. Eop1 mRNA expression is oocyte-specific; Eop1 mRNA is first detected on gestational day 18, decreasing thereafter to minimal levels on the day of birth.  Eop1 mRNA prevalence increases again 48 and 96 h after birth, coinciding with the time of follicular assembly and the initiation of early follicular growth. The specific expression of Eop1 in oocytes and its developmental pattern of expression suggest a role for this novel gene in the regulation of oocyte physiology.
Keywords: Time-course

overall_design: Ovaries from C57Bl/6J were obtained from pups at <24h and 48h after birth. In this strain of mice, assembly of ovarian follicles is initiated 48h after birth, and growth of the newly formed follicles starts shortly thereafter. To analyze differentially expressed mRNA profiles at the time of follicular formation and early follicular growth, we employed a two-dye spotted cDNA microarray containing 8,400 gene probes generated from a mouse NIA 15K gene set (GPL5369), printed in duplicate on glass slides by the Gene Microarray Shared Resource Facility of the Oregon Health & Science University.  RNA samples from 2  (2-1, 2-2, 2-3)  and 4 day-old mice (4-1, 4-2, 4-3) were compared to RNA samples from 0 day-old mice (0-1, 0-2, 0-3, 0-4, 0-8). Each comparison pair was hybridized twice on the same slide (4 experimental replicates) and three RNA sample were used for each age category (3 biological replicates).

first: Sergio

last: Ojeda

first: Elsa

last: De La Chesnaye

first: Bredford

last: Kerr

first: Alfonso

last: Paredes


title: Fasting induces a biphasic adaptive metabolic response in murine small intestine

status: Public on Nov 12 2007

first: Milka

last: Sokolovic

department: Medical Biochemistry

institute: AMC

summary:
Background
The gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting. Results
Intestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gut’s structural components, so that the microarrays’ tissue base remained unaffected. Unsupervised hierarchical clustering of the microarrays revealed that the successive time points separated into distinct branches. Pathway analysis depicted a pronounced, but transient early response that peaked at 12 hours, and a late response that became progressively more pronounced with continued fasting. Early changes in gene expression were compatible with a cellular deficiency in glutamine and metabolic adaptations directed at glutamine conservation, inhibition of pyruvate oxidation, stimulation of glutamate catabolism via aspartate and phosphoenolpyruvate to lactate, and enhanced fatty-acid oxidation and ketone-body synthesis. In addition, the expression of key genes involved in cell cycling and apoptosis was suppressed. At 24 hours of fasting, many of the early adaptive changes abated. Major changes upon continued fasting implied the production of glucose rather than lactate from carbohydrate backbones, a downregulation of fatty-acid oxidation and a very strong downregulation of the electron-transport chain. Cell cycling and apoptosis remained suppressed.
Conclusion
The changes in gene expression indicate that the small intestine rapidly looses mass during fasting to generate lactate or glucose and ketone bodies. Meanwhile, intestinal architecture is maintained by downregulation of cell turnover.
Keywords: fasting response, time course

overall_design:
Male FVB mice (Charles River, Maastricht, The Netherlands) were housed at 20-22°C, 50-60% humidity, a 12 hours light/dark cycle, and food and water ad libitum. At 6 weeks of age, mice were fasted by removing chow for up to 72 hours before sacrifice (n = 6 per group). The animals were kept in metabolic cages to prevent the consumption of beddings and were kept warm with an infrared lamp starting at 24h.
All animals were sacrificed between 9:00 and 10:00 a.m. by cervical dislocation. The small intestine was removed quickly in such a way that adherent tissue remained behind. It was opened longitudinally, rinsed in phosphate-buffered saline, blotted, weighed, snap-frozen in liquid N2, and stored at -80°C. We opted to use extracts of full-thickness intestine for gene-expression profiling, because the epithelial component of the murine small intestine comprises over 70% of its volume The suitability of this strategy is underscored by a recent microarray study of transporters in the mouse intestine [Anderle P et al., BMC Genomics 2005, 6: 69.]. In addition, isolation of enterocytes is time-consuming and, hence, entails a risk of mRNA degradation, while mucosal scraping harvests villi more efficiently than crypts , whereas many mRNAs are most abundant in the crypts.
Total intestinal RNA was extracted from frozen tissue with guanidiniumthiocyanate [Chomczynski P, Sacchi N: Analytical Biochemistry 1987, 162: 156-159.], followed by cesium-chloride centrifugation [Glisin V, Crkvenjakov R BC: Biochemistry 1974, 13: 2633-2637.] to avoid contamination with mucus. The quality of RNA was assessed with the RNA 6000 Nano LabChip® Kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA). Microarray-based quantification of 8 mRNAs with a 1.3-9.2-fold change in expression was validated by qPCR, as described [58]. mRNA concentration was calculated using the LinReg program [59]. In the absence of reverse transcriptase, the signal was < 0.1% of that in its presence for each primer pair (not shown).
Microarrays. The 60-mer Mouse Development (22K) Oligo Microarray G4120A (Agilent) was used. Three arrays per experimental condition were used. Per microarray, 20 μg mRNA, pooled from 2 intestines, was reverse transcribed with Cy3-labelled dCTP (Perkin Elmer, Boston, USA), using the Agilent Fluorescent Direct Label Kit. Cy5-labeled cDNA produced from RNA pooled from 6 fed animals served as the common reference across all arrays (Figure 1A). Hybridized cDNAs were detected with Agilent’s dual-laser microarray slide scanner. The data were retrieved with Agilent’s Feature Extraction software 6.1.1.

last: Sokolović

first: Diederik

last: Wehkamp

first: Aleksandar

first: Jacqueline

last: Vermeulen

first: Lisa

last: Gilhuijs-Pederson

first: Rachel

last: van Haaften

first: Yuri

last: Nikolsky

first: Chris

last: Evelo

first: Antoine

last: van Kampen

first: Theodorus

last: Hakvoort

first: Wouter

last: Lamers


title: The Effect of Lifestyle on Gene Expression in Moroccan Amazighs

status: Public on Nov 15 2007

city: Raleigh

first: Youssef

last: Idaghdour

address: South Gardner Hall

department: Genetics

institute: NCSU

summary:
The human transitions from nomadic to agrarian to urban lifestyles are likely to impact physiology and disease susceptibility.  In order to estimate the magnitude of the impact of lifestyle on genome function, we profiled gene expression in total leukocytes of Moroccan Amazigh from three distinct localities.  Despite great expression heterogeneity in humans, as much as one third of the PBMC transcriptome was found to differ between the localities.
Keywords: Population genomic comparison

overall_design: Total leukocytes were collected in December 2006 from three Moroccan Amazigh populations having three distinct lifestyles. Expression profiles of 10,173 transcripts were obtained for 46 individuals.

first: Greg

middle: C

last: Gibson

first: John

last: Story


title: Formaldehyde treatment with 1.8 mM for 2 h

status: Public on Nov 01 2007

first: Emiko

last: Kitagawa

address: Onogawa 16-1

institute: National Institute of Advanced Industrial Science and Technology

summary:
The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. To obtain varied toxicity-induced gene expression alteration profiles, we examined many kinds of chemicals and compared the gene expression profiles as chemical toxicity each other. Consequently, cells need to be exposed with same experimental cellular condition, semi lethal (IC50), respectively. In the case of formaldehyde (CAS; 50-00-0), the exposure dose was decided as 1.8 mM by growth curve with continuously diluted exposure.  Formaldehyde is one of the aldehydes. This substrate is recognized as a carcinogen and suspected to be gastrointestinal or liver toxicant, immunotoxicant, neurotoxicant, reproductive toxicant, respiratory toxicant, skin or sense organ toxicant.
Keywords: stress response

overall_design: Series containes 3 hybridization results from independent biological samples, and each experiment have high and low power scanned data respectively.

first: Daisuke

last: Yasokawa

first: Hitoshi

last: Iwahashi


title: Genome position and gene amplification - expressional profiling of methotrexate resistant colonies

status: Public on Oct 08 2007

first: Donna

last: Albertson

department: Comprihensive Cancer Center

institute: University of California San Francisco

summary:
We investigated the influence of genome position on propensity to amplify. First, we integrated a mutant form of DHFR into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells. We observed site specific differences in methotrexate sensitivity, organization of amplicons and amplification frequency. One site was uniquely associated with a significantly enhanced propensity to amplify and recurrent amplicon boundaries, possibly implicating a rare folate sensitive fragile site in initiating amplification. Hierarchical clustering of gene expression patterns and subsequent gene enrichment analysis revealed two clusters differing significantly in expression of MYC target genes independent of integration site.
Keywords: Gene amplification, array CGH, chromosomal fragile sites

overall_design: We introduced a mutant form of DHFR (L22F), which confers greater resistance to methotrexate than the wild type (endogenous) gene into HCT116+chr3 cells and isolated independent clones containing DHFR* at different positions in the genome and identified genome sequences flanking the integration site of DHFR* using inverse PCR. For further analysis, we selected only clones, which were considered to have a single insertion of DHFR* by inverse PCR (13 independent insertion sites). The individual insertion site clones were further characterized with respect to genome copy number profiles. To select methotrexate resistant colonies, we exposed cells to a concentration of methotrexate that was three to four times the IC-50 for each integration site. Genomic copy number profiles were obtained for isolated resistant colonies (GSE6262) by using UCSF HumArray platform (GPL4421). Twelve methotrexate resistant colonies (four different integration sites) were selected for microarray analysis of gene expression at the mRNA level. The untreated integration site clone was used as the reference (Cy5 labeled cDNA) for each of the hybridizations with its respective resistant colonies (Cy3 labeled cDNA).  Hybridizations were carried out on arrays of printed long oligonucleotides (70mers) containing 21,000 elements (Operon V2.0, printed in J. David Gladstone Institutes, Genomics Core Laboratory).

first: Pavla

last: Gajduskova

last: Snijders

first: Serena

last: Kwek

first: Ritu

last: Roydasgupta

first: Jane

last: Fridlyand

first: Taku

last: Tokuyasu

first: Daniel

last: Pinkel


title: Dosage compensation in the mouse balances up regulation and silencing of X-linked genes

city: Birmingham

first: Hong

last: Lin

address: Vincent Drive

department: Anatomy

institute: Institute of Biomedical

summary:
Dosage compensation in mammals involves silencing of one X chromosome in XX females, and requires expression, in cis, of Xist silencing RNA. Using microarray analysis to assay expression of X-linked genes in mouse embryonic stem cells, we show that dosage compensation also involves global up-regulation of expression from the active X in both males and females. Up-regulation is complete (ie. 2-fold) only after 2-3 weeks of differentiation. X-linked gene silencing in female cells occurs on a gene-by-gene basis throughout differentiation and includes a sub-group of genes silenced prior to the onset of differentiation and independently of enhanced Xist expression.
Keywords: dosage compensation, gene silencing, Xist RNA, microarray

overall_design: TheNIA 15K cDNA library contains 15,247 cDNA clones with an average insert size of 1.5Kb, which were printed in duplicate. The data presented in the manuscript are derived from the 252 X-linked clones (180 genes) and 6945 autosomal clones (5085 genes) that both correspond to named genes and consistently gave above-background signals with ES cell cDNAs. cDNAs from female and male ES cells at the same stage of differentiation were labelled with Cy3 and Cy5 and equal amounts were co-hybridised to arrays. Each time point have between 2~5 biological replicates.

first: Vibhor

last: Gupta

first: Mathew

last: VerMilyea

first: Francesco

last: Falciani

first: Jeannie

middle: T

last: Lee

first: Laura

last: O’Neill

first: Bryan

last: Turner


title: Zinc pyrithione treatment with 12.6 uM for 2 h

city: Tsukuba

first: Emiko

last: Kitagawa

summary:
The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. In order to evaluate toxicity by newly synthesized or mixture chemicals, toxicity-induced gene expression alteration profiles by known chemicals should be collected. In our study, cells need to be exposed with same experimental cellular condition, semi lethal (IC50), respectively. In the case of zinc pyrithione (CAS; 13463-41-7), the exposure dose was decided as 12.6 uM by growth curve with continuously diluted exposure. Zinc pyrithione is best known for its use in treating dandruff and seborrheic dermatitis. It also has antibacterial properties and is effective against many pathogens from the streptococcus and staphylococcus class. This substrate is suspected to be neurotoxicant and skin or sense organ toxicant.
Keywords: stress response

last: Yasokawa


title: Regulation of gene expression in the liver by naive CD4+ T cells

city: Bethesda

first: Stephen

middle: John

last: Davies

address: 4301 Jones Bridge Road

department: Microbiology and Immunology

institute: USUHS

summary:
We hypothesized that homeostatic interactions between naïve CD4+ T cells and resident cells in the liver generates signals that modulate gene expression in this organ. To test this hypothesis, we identified genes that are regulated by the presence of naïve CD4+ T cells by using a whole genome microarray to compare transcript levels in liver tissue from non-infected RAG-1-/-, OT-II/RAG-1-/- and wild type mice (Fig. 3). Using this approach, we identified 175 genes that were differentially expressed only in the presence of naïve CD4+ T cells.
Keywords: Genotype comparison

overall_design: Livers from naïve female wild type C57BL/6, RAG-/-, and OT-II/RAG-/- (N=9 for each genotype) mice were pooled into groups of three and prepared for cDNA microarray analysis.  Briefly, RNA was isolated via RNAzol (Tel-Test, Friendswood, TX) and the RNeasy protocol (Qiagen) and analyzed for purity and concentration on a NanoDrop® ND-1000 Spectrophotometer (Wilmington, DE). cDNA was prepared from two 30 µg aliquots of each pooled sample and labeled with either Cy3 or Cy5 fluorescent probes.  One 30 µg aliquot from each pool was used to create a background control pool, while the second aliquot was used as the comparative sample. For further isolation and labeling protocol details please refer to http://www.niaid.nih.gov/dir/services/rtb/microarray/protocols.asp.  Samples were hybridized as described by Schaupp (9) to Mmbe custom arrays manufactured by the NIAID Microarray Facility.  Further information about these arrays can be found at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL1057.  Hybridizations were performed in triplicate. Images were scanned by GenePix4000B Scanner (Axon Instruments/Molecular Devices, Sunnyvale, CA) and analyzed using the mAdb program (http://madb.niaid.nih.gov/).  Signals were calculated as mean intensity – median background. Data were analyzed using Significance Analysis of Microarrays (SAM), version 2.11 (Stanford University) and student’s T tests (EXCEL, with P values <0.05 considered significant) to identify only those genes that exhibited differential expression in RAG-1-/- liver tissue compared to both OT-II/RAG-1-/- and wild type liver tissue (i.e. genes differentially expressed in RAG-1-/- liver tissue compared to either OT-II/RAG-1-/- or wild type alone were not considered specific to the presence of naïve CD4+ T cells).

first: Erika

middle: W

last: Lamb

first: John

last: Pesce

first: Emily

last: Crow

first: Sean

middle: K

last: Maynard

first: Thomas

last: Wynn

first: Brian

last: Schaefer


title: Gene Regulation by HDAC7 in Thymic Selection

status: Public on Nov 04 2007

city: Novato

first: Eric

last: Verdin

address: 8001 Redwood Blvd

institute: Buck Institute for Research on Aging

summary:
Histone deacetylase 7 (HDAC7) is highly expressed in CD4+/CD8+ thymocytes and functions as a signal-dependent repressor of gene transcription during T cell development. In this study, we express HDAC7 mutant proteins in a T cell line and use DNA microarrays to identify transcriptional targets of HDAC7 in T cells. Gene expression changes are compared to differential gene expression profiles associated with positive and negative thymic selection.  This analysis reveals that HDAC7 regulates an extensive set of genes that are differentially expressed during both positive and negative thymic selection. Many of these genes play important functional roles in positive and negative selection, primarily via coupling between antigen receptor and downstream signaling events.
Keywords: microarray gene expression profiling, comparison of perturbation of HDAC7 gene function with differential expression during thymic selection

overall_design: DO11.10 cells were transduced with different viruses encoding mutants of HDAC7, MEF2D, or Nur77. All microarrays were hybridized with untreated DO11.10 cells in one channel and transdced/treated cells in the other. Hybridizations are done with 4-12 replicates. DO11.10 TCR-transgenic thymocytes were also compared to MHC-deficient thymocytes and DO11.10 TCR-transgenic thymocytes from animals that had been injected with antigenic peptide at different timepoints prior to harvest of thymocytes.

first: Herbert

last: Kasler


title: human prostate and breast epithelial cell lines under EZH2 adenovirus overexpression

city: Chicago

first: Jindan

last: Yu

state: IL

address: 303 E. Superior St.

department: Medicine - Hem/Onc

institute: Northwestern University

summary:
Transcriptional profiling of human prostate and breast epithelial cells with a time course analysis of EZH2 overexpression of cells at 3, 6, 12, 24, 48, and 72 hrs respectively.
Keywords: Genetic modification

overall_design: timse-course experiment, adenovirus EZH2 infected cells vs. vector infected cells at a series of time points.

first: Arul

last: Chinnaiyan


title: human prostate and breast epithelial cell lines with EZH2 siRNA interference

summary:
Transcriptional profiling of human prostate and breast epithelial cells comparing control siRNA-treated with EZH2-siRNA treated
Keywords: Genetic modification

overall_design: Two-condition experiment, fluorescent control siRNA vs EZH2 siRNA-transfected cells. Biological replicates: 3 control-transfected, 3 EZH2-transfected, independently grown and harvested.


title: Methanol treatment with 5 % for 2 h

summary:
The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. To obtain varied toxicity-induced gene expression alteration profiles, we examined many kinds of chemicals and compared the gene expression profiles as chemical toxicity each other. Consequently, cells need to be exposed with same experimental cellular condition, semi lethal (IC50), respectively. In the case of methanol (CAS; 67-56-1), the exposure dose was decided as 5 % by growth curve with continuously diluted exposure.  Methanol is one of the alcohols, and suspected to be developmental, gastrointestinal or liver, kidney, neuro-, respiratory toxicant.
Keywords: stress response

first: Daisuke

first: Hitoshi

last: Iwahashi