title: Identification of the WhiB5 regulon in Mycobacterium tuberculosis H37Rv

status: Public on Aug 15 2011

city: Padova

first: Stefano

last: Casonato

address: via A. Gabelli, 63

department: Histology, microbiology and medical biotechnologies

country: Italy

institute: University of Padova

summary: We used DNA microarrays to define the M. tuberculosis whiB5 regulon and to study the variation of the global transcription profile in response of its induction. For this purpose we constructed a M. tuberculosis strain in which whiB5 was transcribed from an pristinamycin-inducible promoter and its transcriptional profile was compared on DNA microarray with the transcriptional profile of its control strain (empty vector-containing clone). WhiB5 induction for 15’ resulted in the induction of 26 genes, while induction for 3 h resulted in the induction of 58 genes. Interestingly, all of the 26 genes induced after 15 minutes of incubation with pristinamycin were still induced after 3 h of incubation. No repressed genes were identified. Among the induced genes we found the alternative sigma factor SigM and at least 12 genes previously shown to be regulated by this sigma factor, including those encoding the ESX-4 type VII secretion system. Interestingly, also the genes encoding ESX-2 type VII secretion system were strongly induced together with an operon probably involved in hydrogen metabolism in anaerobiosis. 10 representative genes were chosen from the microarray experiments and their expression was measured by quantitative RT-PCR using sigA as invariant internal control. In support to the gene expression profiling data, the mRNA levels of all selected genes was clearly higher in the whiB5 mutant strain relative to control.

overall_design: We compared the global gene expression of the whiB5 overexpressing strain and the control strain of Mtb in different conditions: after 15 min of induction or after 3 hours of induction. Hybridizations were performed using RNA extracted from three different biological samples. Each sample was hybridized twice through swap labeling of the respective cDNAs.

first: Riccardo

last: Manganelli


title: Genome-scale epigenetic reprogramming during epithelial to mesenchymal transition

status: Public on Jul 03 2011

city: Baltimore

first: Andrew

last: Feinberg

state: MD

address: 855 N. Wolfe Street

department: Center for Epigenetics and Department of Medicine

country: USA

institute: Johns Hopkins University

type: Genome binding/occupancy profiling by genome tiling array

summary: Epithelial to mesenchymal transition (EMT) is an extreme example of cell plasticity, important for normal development, injury repair, and malignant progression. Widespread epigenetic reprogramming occurs during stem cell differentiation and malignant transformation, but EMT-related epigenetic reprogramming is poorly understood. Here we investigated epigenetic modifications during TGF-β-mediated EMT. While DNA methylation was unchanged during EMT, we found global reduction of the heterochromatin mark H3-lys9 dimethylation (H3K9Me2), increase of the euchromatin mark H3-lys4 trimethylation (H3K4Me3), and increase of the transcriptional mark H3-lys36 trimethylation (H3K36Me3). These changes were largely dependent on lysine-specific deaminase-1 (LSD1), and LSD1 loss-of-function experiments showed marked effects on EMT-driven cell migration and chemoresistance. Genome-scale mapping revealed that chromatin changes were largely specific to large organized heterochromatin K9-modifications (LOCKs), suggesting that EMT is characterized by reprogramming of specific chromatin domains across the genome.

overall_design: Chromatin immunoprecipitation (ChIP) was performed with antibodies against H3K9Me2 (Abcam, ab1220), H3K36Me3 (ab9050), and H3K4Me3 (ab8580) on native (unfixed) chromatin isolated from fully differentiated mouse AML12 cells (confluent, serum starved for 48hrs) either treated with TGF-β for 36hrs to induce EMT or not treated with TGF-β (0hrs, differentiated AML12 cells). DNA purified from these samples was then either whole-genome amplified (H3K36Me3 and H3K4Me3) and hybridized or directly (H3K9Me2) hybridized to NimbleGen 2.1M economy whole-genome tiling arrays #2 (listed below as array 1) and #3 (listed below as array 2), which cover mouse chromosomes 4-14. For each sample, the immunoprecipitated DNA (IP) and the input (control) DNA were hybridized to the arrays, and the IP is normalized to the input. There are 16 total samples listed below. There are 8 sample for H3K9Me2 (TGF-β and no TGF-β for arrays 1 and 2, done in replicate for a total of 8). There are 4 samples for H3K36Me3 (TGF-β and no TGF-β done on array 1 and array 2). There are 4 total samples for H3K4Me3 (TGF-β and no TGF-β done on array 1 and array 2).

first: Oliver

middle: G

last: McDonald

first: Hao

last: Wu

first: Winston

last: Timp

first: Akiko

last: Doi

middle: P


title: Chip-chip from mouse olfactory epithelium (OE) and liver with antibodies against H3K9me3 and H4K20me3

status: Public on May 03 2011

city: San Francisco

first: Stavros

last: Lomvardas

state: CA

institute: Univesity of California San Francisco

summary:
The Olfactory Receptor (OR) genes are specifically expressed in the MOE in a monogenic and monoallelic fashion. Only 1 out of 2800 alleles is expressed in each olfactory sensory neuron in mice.
ChIP-chip from mouse olfactory epithelium (OE) and liver for H3K9me3 and H4K20me3 revealed that the ORs are highly enriched for these modifications in OE but not in liver.

overall_design: 24 samples were analyzed (20 from OE and 4 from liver) with matching inputs as controls. For the OE samples, there were 2-3 replicates for each array.


title: Gene expression array assay of Lsh WT and Knockout MEF cells

status: Public on May 19 2011

city: Frederick

first: Jigui

last: Shan

address: 430 Miller Dr

institute: NCI-Frederick

summary: DNA methylation is critical for normal development and plays important roles in genome organization and transcriptional regulation. Although DNA methyltransferases have been identified, the factors that establish and contribute to genome-wide methylation patterns remain elusive. Here, we report a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences. We provide evidence that Lsh also controls genome-wide cytosine methylation at nonrepeat sequences and relate those changes to alterations in H4K4me3 modification and gene expression. Deletion of Lsh alters the allocation of cytosine methylation in chromosomal regions of 50 kb to 2 Mb and, in addition, leads to changes in the methylation profile at the 5′ end of genes. Furthermore, we demonstrate that loss of Lsh promotes—as well as prevents—cytosine methylation. Our data indicate that Lsh is an epigenetic modulator that is critical for normal distribution of cytosine methylation throughout the murine genome.

overall_design: We used microarrays to detail a correlation between cytosine methylation of murine genome modulated by Lsh at non-repetitive sequence and corresponding gene expression.

first: Alika

last: Maunakea

first: Anney

last: Che

first: Victorino

last: Briones

first: Eunice

middle: Y

last: Lee

first: Theresa

last: Geiman

first: Jiaqiang

last: Huang

first: Robert

last: Stephens

middle: M

last: Leighty

first: Keji

last: Zhao

first: Kathrin

last: Muegge


title: Study the effect of mesenchymal stem cells on isolated cortical neurons

status: Public on May 01 2011

city: Athens

first: Vivian

last: Tseveleki

address: 127 Vasilissis Sophias Avenue

department: Molecular Genetics

country: Greece

institute: Hellenic Pasteur Institute

summary: Primary cortical neurons were isolated from E15 mice and after 6 days in vitro were untreated or treated for 24 h with mesenchymal stem cell conditioned medium. Neuron gene expression was profiled and compared between the two different conditions (neurons and neurons+MSC cm) to investigate the molecular mechanisms of MSC neuroprotection. Mesenchymal stem cells (MSC) promote functional recovery in experimental models of central nervous system (CNS) pathology and are currently being tested in clinical trials for stroke, multiple sclerosis and CNS injury. Their beneficial effects are attributed to the activation of endogenous CNS protection and repair processes as well as immune regulation but their mechanisms of action are poorly understood. Here we investigated the neuroprotective effects of mouse MSC in rodent MSC-neuron co-cultures and mice using models of glutamate excitotoxicity. A 24 hr pre-culture of mouse primary cortical neurons with MSC protected them against glutamate (NMDA) receptor-induced death and conditioned medium from MSC (MSC CM) was sufficient for this effect. Protection by MSC CM was associated with reduced mRNA levels of genes encoding NMDA receptor subunits, and increased levels for genes associated with non-neuronal and stem cell types, as shown by RT-PCR and cDNA microarray analyses. Changes in gene expression were not associated with alterations in cell lineage representation within the cultures. Further, MSC CM-mediated neuroprotection in rat retinal ganglion cells was associated with reduced glutamate-induced calcium influx. The adoptive transfer of EGFP+MSC in a mouse kainic acid epilepsy model also provided neuroprotection against glutamate excitotoxicity in vivo, as shown by reduced neuron damage and glial cell activation in the hippocampus. These results show that MSC mediate direct neuroprotection by reducing neuronal sensitivity to glutamate receptor ligands and altering gene expression, and suggest a link between the therapeutic effects of MSC and the activation of cell plasticity in the damaged CNS.

overall_design: In vitro cultures primary cortical neurons from mice were protected from glutamate excitotoxicity when pre-treated with MSC cm. Global gene expression changes induced in neurons before and after treatment with MSC cm were investigated using a cDNA spotted macroarray filter. Two samples were analyzed in duplicate: neurons alone (untreated) and neurons+MSC cm.

first: Lesley

last: Probert


title: Early Origins Meta-peak generation

status: Public on Mar 16 2011

city: Toronto

first: DCC

last: modENCODE

state: Ontario

address: MaRS Centre, South Tower, 101 College Street, Suite 800

country: Canada

institute: Ontario Institute for Cancer Research

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
modENCODE_submission_3441
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
We will precisely identify sequence elements that direct DNA replication by using chromatin immunoprecipitation of known replication initiation complexes.  These experiments will be conducted in multiple cell types and developmental tissues.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: Output file represents the union of the coordinates of peak data from various experiments peak calls. These unioned areas, referred to as meta-peaks, cover any genomic coordinate that has been experimentally shown to be capable of sustaining a peak.

first: David

last: MacAlpine

first: Terry

last: Orr-Weaver

first: Jarred

last: Norman

first: Sharon

last: Li

first: Joseph

last: Prinz


title: Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function   in transcriptional regulation in mouse embryonic stem cells

status: Public on Mar 30 2011

city: Boston

first: Hao

last: Wu

state: Massachusetts

address: 149G Warren Alpert Building, 200 Longwood Avenue

department: Genetics

institute: Harvard Medical School/HHMI

type: Methylation profiling by genome tiling array

summary: Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC).  While 5mC has been studied extensively, little is known about the distribution and function of 5hmC.  Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells revealed suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators.  Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells and supports its dual function in regulating gene expression.

overall_design: Genomic DNA extracted from control (Con) or Tet1 knockdown (KD) mouse ES cells was immunoprecipitated with 5-hydroxymethylcytosine (5hmC) antibodies and analyzed by NimbleGen 2.1M mouse whole genome tiling microarrays (a 4-array set covering the entired non-repetitive portion of mouse genome).  Whole cell extract (WCE) was used as input controls in IP/input experiments.

first: Ana

last: D'Alessio

first: Shinsuke

last: Ito

first: Zhibin

last: Wang

first: Yi

last: Sun

first: Kairong

last: Cui

last: Zhang


title: Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells (ChIP-chip and MeDIP-chip)

summary: Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. Recent demonstration that members of the Ten-eleven translocation (Tet) family proteins can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell self-renewal and maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), here we show that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite a general increase in levels of DNA methylation at Tet1 binding-sites, Tet1 depletion does not lead to down-regulation of all the Tet1 targets. Interestingly, while Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also required for repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators.

overall_design: Chromatin or genomic DNA extracted from control (Con) or Tet1 knockdown (KD) mouse ES cells was immunoprecipitated with indicated antibodies and analyzed by NimbleGen 2.1M mouse whole genome tiling microarrays (a 4-array set covering the entired non-repetitive portion of mouse genome).  Whole cell extract (WCE) was used as input controls in IP/input experiments. In IP/IP experiments, immunoprecipitated DNA from Con KD and Tet1 KD ES cells was directly compared on the same microarrays.

first: Kai

last: Xia

last: Zhao


title: ChIP-chip mapping of twist, dorsal, snail, weckle in Drosophila embryos

status: Public on Feb 28 2011

city: Los Altos

first: Manoj

last: Samanta

address: 2240 Homestead Court #309

institute: Systemix Institute

summary:
We performed ChIP-chip analysis using sheared chromatin
isolated from Drosophila embryos of 1-3 hours in age.
Keywords: high-resolution tiling array, drosophila, twist, dorsal, snail, weckle

overall_design: ChIP-chip of twist, dorsal, snail, and weckle

first: Anil

last: Ozdemir

first: Katherine

last: Fisher

first: Shirley

last: Pepke

first: Leslie

last: Dunipace

first: Kenneth

last: McCue

first: Lucy

last: Zeng

first: Nobuo

last: Ogawa

first: Barbara

last: Wold

first: Angelike

last: Stathopoulos


title: Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy

status: Public on Mar 01 2011

city: Rochester

first: Jeff

last: Nie

state: MN

address: 200 First St. SW

institute: Mayo Clinic

type: Genome variation profiling by genome tiling array

summary: Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a novel approach to gene therapy. Yet it is unknown whether selective pressures during prolonged culture and multiple clonal events would introduce a mutational load incompatible with therapeutic use. Here we begin to assess whether the mutational load of gene-corrected iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected following standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine point mutations in protein-coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base pair change, no additional mutations or copy number variation were identified in iPS cells following the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.

overall_design:
Array comparative genomic hybridization (aCGH) were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette.
Human CGH 2.1M Whole-Genome Tiling v2.0D Array plus 4633 custom-designed probes which cover the gene targeting construct and the reprogramming plasmid vectors. Comparison of patient iPS cell with full skin fibroblast cells was performed on this design.

first: Sara

middle: E

last: Howden


status: Public on May 05 2011

city: Stanford

first: Rami

last: Rauch

address: 300 Pasteaur Dr

institute: Stanford U

summary:
Publication title:  Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver
To investigate the role of DNA methylation during human development, we developed Methyl-seq, a method that assays DNA methylation at more than 90,000 regions throughout the genome.  Performing Methyl-seq on human embryonic stem cells (hESCs), their derivatives and human tissues allowed us to identify several trends during hESC and in vivo liver differentiation.  First, differentiation results in DNA methylation changes at a minimal number of assayed regions, both in vitro and in vivo (2-11%).  Second, in vitro hESC differentiation is characterized by both de novo methylation and demethylation, whereas in vivo fetal liver development is characterized predominantly by demethylation.  Third, hESC differentiation is uniquely characterized by methylation changes specifically at H3K27me3-occupied regions, bivalent domains and low-density CpG promoters (LCPs) suggesting that these regions are more likely to be involved in transcriptional regulation during hESC differentiation.  Although both H3K27me3-occupied domains and LCPs are also regions of high variability in DNA methylation state during human liver development, these regions become highly unmethylated, which is a distinct trend from that observed in hESCs.  Taken together, our results indicate that hESC differentiation has a unique DNA methylation signature that may not be indicative of in vivo differentiation.
Keywords: Methyl-seq, hESC differentiation

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

overall_design: Illumina gene expression beadchips of human ES cell lines, ES-derived cells, and normal liver (15 samples).

first: Alayne

middle: L

last: Brunner

first: David

middle: S

last: Johnson

first: Si

middle: W

last: Kim

first: Anton

last: Valouev

first: Timothy

last: Reddy

first: Norma

middle: F

last: Neff

first: Elizabeth

last: Anton

first: Catherine

last: Medina

first: Loan

last: Nguyen

first: Eric

last: Chiao

first: Chuba

middle: B

last: Oyolu

first: Gary

last: Schroth

first: Devin

last: Absher

first: Julie

middle: C

last: Baker

first: Richard

last: Myers


title: Evidence for Zoonotic Transmission of a Novel Adenovirus Associated with a Fulminant Pneumonia Outbreak in a Titi Monkey Colony

status: Public on Feb 05 2011

first: Charles

last: Chiu

address: 185 Berry Street, Box 0134

department: Laboratory Medicine

institute: University of California, San Francisco

summary: We used the Virochip microarray to detect and discover a novel adenovirus, TMAdV (titi monkey adenovirus).  We used custom-commercial microarrays from Agilent Technologies.  The microarray platform GPL11662 consists of 62,976 probes, of which 19,058 probes are 70-mer viral oligonucleotides (the union of the V3 and V4 platforms -- please see Wang, et al., PNAS, 2002; Chiu, et al., Clin Infect Dis, 2006; Chiu, et al., PNAS, 2008).

sample_organism:
Homo sapiens
Plecturocebus cupreus

overall_design: For this study, 4 experimental Virochip microarrays derived from tissue and swabs from affected titi monkeys were analyzed.  We also used 18 Virochip microarrays derived from sera from healthy random human blood donors as a negative background set for Z-score analysis (see Chiu, et al., Clin Infect Dis, 2006).  Raw Agilent microarray .txt files are background normalized and the 19,058 70-mer viral probes are then selected and displayed as columns in the processed data table.

first: Eunice

last: Chen


title: Metastatic potential of CNE-2 cells

status: Public on Feb 14 2011

city: Grand Rapids

first: Karl

last: Dykema

state: MI

address: 333 Bostwick Ave. NE

institute: Van Andel Institute

summary:
Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential.  From genomic expression profiles comparing clones derived from the NPC cell line CNE-2, serglycin (SRGN) was identified as one of the most up-regulated genes in the high-metastasis clone.  Serglycin protein was secreted by the high-metastasis clone, but not by any of the low-metastasis clones.  Suppression of serglycin by shRNA diminished serglycin secretion and subsequently inhibited the migration and invasion of high-metastasis clone, and also reduced its metastasis rate in vivo.  Overexpression of serglycin in low-metastasis cells resulted in an increased metastasis rate in vivo.  Moreover, secreted serglycin promoted cellular motility in the wild-type low-metastasis cells. Interestingly, suppression of serglycin reduced the protein level of vimentin but did not influence the level of E-cadherin in high-metastasis clone. Proliferation was not influenced by serglycin in both high-and low-metastasis clones. Clinically, serglycin expression was significantly elevated in liver metastases from NPC relative to its expression in primary tumors.  The prognostic value of serglycin was evaluated by immunohistochemical staining of tissue microarrays of NPC tissues from 263 patients, followed by multivariate analyses.  A high level of serglycin expression in primary NPC was found to be an independent unfavorable indicator for distant-metastasis-free survival and disease-free survival.  In summary, serglycin regulates NPC metastasis via autocrine and paracrine means without influencing proliferation, and it serves as a prognostic indicator of metastasis-free survival and disease-free survival for NPC patients.

Keywords: Gene Expression experiment

overall_design:
High-metastasis clone (S18) and low-metastasis clones (S22 and S26) together with their parental line CNE-2 were subjected for gene expression profiling to identify the genes highly expressed in high-metastasis clone.
To explore the molecular mechanism(s) underlying this important biological behavior, we performed genomic expression profiling of these four cell lines from in vitro cultured cells, as well as in vivo xenograft tumors generated in nude mice by subcutaneous injection of the cells into the BALB/c (nu/nu) female mice.  Briefly, 1 × 10^5 cells were subcutaneously injected into the left flank of a nude mouse, and the tumors were collected when their volume was 150-250 mm^3.


title: Analysis of mRNA levels of GPCR and GPCR-related signaling proteins in the HEK293 cell line

status: Public on Feb 01 2011

city: Notre Dame

first: Jacqueline

last: Lopez

state: Indiana

address: 019 Galvin Life Science Center

department: Biology

institute: University of Notre Dame

summary: Measurements of mRNA levels for non-chemosensory GPCRs, G proteins and selected G protein targets.

overall_design: HEK293 cell line, 4 replicates. Overall data set includes mRNA levels for ~24,000 genes.

first: Brady

middle: K

last: Atwood

first: Jaqueline

first: James

last: Wager-Miller

first: Ken

last: Mackie

first: Alex

last: Straiker


title: Empirical Annotation of the Daphnia pulex genome; Experiment A

type: Expression profiling by genome tiling array

summary: Experiments conducted on this tiling array are used to (1) validate the frozen gene sets of the current genome annotation, (2) improve the predicted gene structures by empirically determining UTRs and intron-exon boundaries, identifying missing upstream, internal, and downstream exons and alternative transcripts, (3) propose gene structure models in transcribed regions containing no predicted genes and (4) delineate transcriptionally active regions of the genome from intergenic, intronic and genic regions. Signal to background ratios were determined by first calling probes that fluoresced at intensities greater than 99% of the random probes’ signal intensities; therefore only 1% of fluorescing experimental probes should be false positives.

overall_design: We conducted two-color competitive hybridizations that measure differential expression from three replicates, each using RNA from independent biological extractions. Transcriptional active regions (TARs) were defined by stringing together overlapping probes showing fluorescence above a 1% false positive rate (FPR). Positive probes were joined into a TAR if they were adjacent (maxgap=0, no intermittent non-positive probe) and a TAR’s length had to be at least 45 bp (minrun=45, mid-point first positive probe to mid-point last positive probe, resulting in at least 3 adjacent positive probes for a TAR).The data analysis to measure differential expression of genes and of unannotated TARs was performed using the statistical software package R and Bioconductor with additions and modifications. The signal distributions across chips, samples and replicates were adjusted to be equal according to the mean fluorescence of the random probes on each array. All probes including random probes were quantile-normalized across replicates. Expression-level scores were assigned for each predicted gene based on the median log2 fluorescence over background intensity of probes falling within the exon boundaries.

first: John

last: Colbourne

first: Jeong-Hyeon

last: Choi


title: Analysis of mRNA levels of GPCR and GPCR-related signaling proteins in 3 mouse cell lines: AtT20, BV2 and N18

overall_design: Three mouse cell lines (AtT20, BV2 and N18), 4 replicates each. Overall data set includes mRNA levels for ~24,000 genes.


title: Uncovering Genes and Regulatory Pathways Related to Urinary Albumin Excretion in Mice

city: Bar Harbor

first: Rachael

last: Hageman

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

summary: Identifying the genes underlying quantitative trait loci (QTL) for disease has proven difficult, mainly due to the low resolution of the approach and the complex genetics involved. However, recent advances in bioinformatics and the availability of genetic resources now make it possible to narrow the genetic intervals and test candidate genes. In addition to identifying the causative genes, defining the pathways that are affected by these QTL is of major importance as it can give us insight into the disease process and provide evidence to support candidate genes. In this study we mapped three significant and one suggestive QTL on Chromosomes (Chrs) 1, 4, 15, and 17, respectively, for increased albumin excretion (measured as albumin-to-creatinine ratio) in a cross between the MRL/MpJ and SM/J mouse inbred strains. By combining data from several sources and by utilizing gene expression data, we identified Tlr12 as a likely candidate for the Chr 4 QTL. Through the mapping of 33,881 transcripts measured by microarray on kidney RNA from each of the 173 male F2 animals, we identified several downstream pathways associated with these QTL. Among these were the glycan degradation, leukocyte migration, and antigen presenting pathways. We demonstrate that by combining data from multiple sources, we can identify not only genes that are likely to be causal candidates for QTL, but also the pathways through which these genes act to alter phenotypes. This combined approach provides valuable insights into the causes and consequences of renal disease.

overall_design:
Female MRL/MpJ (MRL) mice were crossed with male SM/J (SM) mice; their progeny were intercrossed to produce 371 F2 animals.  Only 173 F2 males were used for this study.

This submission represents the expression profiling component of the study.

first: Magalie

last: Leduc

first: Christina

middle: R

last: Caputo

first: Shring-Wern

last: Tsiah

middle: A

last: Churchill

first: Beverly

last: Paigen

first: Ron

last: Korstanje


title: Singleminded-2s (Sim2s) promotes delayed Involution of the Mouse Mammary Gland through suppression of Stat3 and NFkB

status: Public on Feb 03 2011

city: College Station

first: Weston

last: Porter

state: Texas

department: Integrative Biosciences

institute: Texas A&M University

summary: Post-lactational involution of the mammary gland provides a unique model to study breast cancer susceptibility and metastasis.  We have shown that the short isoform of Singleminded-2 (Sim2s), a bHLH/PAS transcription factor, plays a role in promoting lactogenic differentiation, as well as in maintaining mammary epithelial differentiation and malignancy.  Sim2s is dynamically expressed during mammary gland development, with expression peaking during lactation, and decreasing in early involution.  To determine the role of Sim2s in involution, we used transgenic mice expressing Sim2s under the mouse mammary tumor virus (MMTV-Sim2s) promoter.  Over-expression of Sim2s in the mouse mammary gland resulted in delayed involution, indicated by a lower proportion of cleaved caspase-3 positive cells and slower re-establishment of the mammary fat pad.  Immunohistochemical and quantitative RNA analysis showed a decrease in apoptotic markers and inflammatory response genes, and an increase in anti-apoptotic genes, which were accompanied by inhibition of signal transducer and activator of transcription 3 (Stat3) activity.  Microarray analysis confirmed that genes in the Stat3 signaling pathway were repressed by Sim2s expression, along with NFκB and other key pathways involved in mammary gland development.  Multiparous MMTV-Sim2s females displayed a more differentiated phenotype compared to wild-type controls, characterized by enhanced β-casein expression and alveolar structures. Together, these results suggest a role for Sim2s in the normal involuting gland, and identify potential down-stream pathways regulated by Sim2s.

overall_design: Gene expression was measured in 6 mammary gland samples using CodeLink Mouse Whole Genome microarrays.  3 samples were from wild-type mice harvested at 72 hours post weaning and 3 samples were MMTV-Sim2s mice harvested at 72 hours post weaning.

first: Kelly

last: Scribner

first: Elizabeth

last: Wellberg

first: Richard

last: Metz


title: Empirical Annotation of the Daphnia pulex genome; Experiment C

first: Jacqueline

last: Lopez

overall_design: We conducted two-color competitive hybridizations that measure differential expression from three replicates, each using RNA from independent biological extractions. Transcriptional active regions (TARs) were defined by stringing together overlapping probes showing fluorescence above a 1% false positive rate (FPR). Positive probes were joined into a TAR if they were adjacent (maxgap=0, no intermittent non-positive probe) and a TAR’s length had to be at least 45 bp (minrun=45, mid-point first positive probe to mid-point last positive probe, resulting in at least 3 adjacent positive probes for a TAR). Transcriptional active regions (TARs) were defined by stringing together overlapping probes showing fluorescence above a 1% false positive rate (FPR).  The data analysis to measure differential expression of genes and of unannotated TARs was performed using the statistical software package R and Bioconductor with additions and modifications. The signal distributions across chips, samples and replicates were adjusted to be equal according to the mean fluorescence of the random probes on each array. All probes including random probes were quantile-normalized across replicates. Expression-level scores were assigned for each predicted gene based on the median log2 fluorescence over background intensity of probes falling within the exon boundaries.


title: Empirical Annotation of the Daphnia pulex genome; Experiment B

summary: Experiments conducted on this tiling array are used to (1) validate the frozen gene sets of the current genome annotation, (2) improve the predicted gene structures by empirically determining UTRs and intron-exon boundaries, identifying missing upstream, internal, and downstream exons and alternative transcripts, (3) propose gene structure models in transcribed regions containing no predicted genes and (4) delineate transcriptionally active regions of the genome from intergenic, intronic and genic regions. Signal to background ratios were determined by first calling probes that fluoresced at intensities greater than 99% of the random probes’ signal intensities; therefore, only 1% of fluorescing experimental probes should be false positives.

overall_design: We conducted two-color competitive hybridizations that measure differential expression from three replicates, each using RNA from independent biological extractions. Transcriptional active regions (TARs) were defined by stringing together overlapping probes showing fluorescence above a 1% false positive rate (FPR). Positive probes were joined into a TAR if they were adjacent (maxgap=0, no intermittent non-positive probe) and a TAR’s length had to be at least 45 bp (minrun=45, mid-point first positive probe to mid-point last positive probe, resulting in at least 3 adjacent positive probes for a TAR). The data analysis to measure differential expression of genes and of unannotated TARs was performed using the statistical software package R and Bioconductor with additions and modifications. The signal distributions across chips, samples and replicates were adjusted to be equal according to the mean fluorescence of the random probes on each array. All probes including random probes were quantile-normalized across replicates. Expression-level scores were assigned for each predicted gene based on the median log2 fluorescence over background intensity of probes falling within the exon boundaries.


title: N2_Worm_Embryo_mRNA_Expression

status: Public on Nov 18 2010

first: DCC

last: modENCODE

summary:
modENCODE_submission_2762
This submission comes from a modENCODE project of Steven Henikoff. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: poly A+ RNA was isolated and used to synthesize single-stranded cDNA probes.  Labeled cDNA and labeled genomic DNA were hybridized to NimbleGen microarrays (2-color hybridizations).  These experiments are used as controls for nucleosome salt fractionations.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: tiling array analysis. BIOLOGICAL SOURCE: Strain: N2(official name : N2 genotype : wild type genotype : DR subclone of DB original (Tc1 pattern I) ); Developmental Stage: Young Adult; Genotype: wild type; Sex: Hermaphrodite;  NUMBER OF REPLICATES: 2;  EXPERIMENTAL FACTORS: Developmental Stage Young Adult; strain (organism) N2

first: Steven

last: Henikoff

first: Jorja

first: Kami

last: Ahmad


title: Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A

status: Public on Jan 03 2011

city: Mannheim

first: Carsten

last: Sticht

address: Theodor-Kutzer-Ufer

department: ZMF

country: Germany

institute: University Heidelberg

summary: Analysis of bone marrow derived macrophages (BMDM) incubated with dexamethasone&IL4 (Dexa+IL4), B16F1 tumor conditioned medium (cmB16), and B16F1 tumor conditioned medium supplemented with dexamethasone&IL4 (cmB16+dexa+IL4). Results allow detection of genes that require synergistic stimulation of tumor factors and Th2 cytokines.

overall_design: Bone marrow cells were isolated from the tibias and femurs of C57BL/6 mice. M-CSF at a concentration of 30 ng/ml was added to the culture medium [DMEM, 10% FCS, and penicillin/streptomycin]. After 4 days, the culture medium was replaced by fresh DMEM or B16F1 conditioned medium. As indicated, IL-4 (10 ng/ml), dexamethasone (5 × 10−7M), Iwas added. Cells were harvested 3 days later for RNA isolation.

first: Astrid

last: Schmieder

first: Kai

last: Schledzewski

first: Julia

last: Michel

first: Jan

last: Tuckermann

first: Lydia

last: Tome

first: Cleopatra

last: Gkaniatsou

last: Nicolay

first: Alexandra

last: Demory

first: Jörg

last: Faulhaber

last: Kzhyshkowska

first: Cyrill

last: Géraud

first: Sergij

last: Goerdt


title: Role of DNA methylation in FOXA1 cell type-specific recruitment to chromatin

status: Public on Jan 06 2011

city: Lille

first: Jérôme

last: Eeckhoute

address: Bâtiment J&K, Faculté de Médecine de Lille-Pôle Recherche, Boulevard du Professeur Leclerc

country: France

institute: CNRS

summary:
CTCF, H2AFZ and FOXA1 genomic recruitment sites were determined using ChIP-chip while MeDIP-chip was used to monitor DNA methylation levels. Amplified and labeled DNA was hybridized to Affymetrix tiling arrays covering human chromosomes 8, 11 and 12. Cells used in this study are: MCF7 breast cancer cells, LNCaP prostate cancer cells, MDA-MB-231 breast cancer cells stably transfected with a FOXA1 expression vector (MDA231-FOXA1) or the empty control plasmid (MDA231-CTRL).

H3K4me2 genomic distribution was determined using ChIP-chip. Amplified and labeled DNA was hybridized to Affymetrix tiling arrays covering human chromosomes 8, 11 and 12. Cells used in this study are MDA-MB-231 breast cancer cells stably transfected with a FOXA1 expression vector (MDA231-FOXA1) or the empty control plasmid (MDA231-CTRL).

overall_design: Cells were grown in full media and harvested to prepare genomic DNA used in MeDIP-chip (Weber et al. 2005). For ChIP-chip, cells were cross-linked and processed as in (Carroll et al. 2005). DNA was hybridized to Human Affymetrix tiled array F.


title: Dm_y[1]cn[1] bw[1] sp[1]_embryo_4-6h_Poly-Ap_p200_409-410-411_7bp

last: modENCODE

summary:
modENCODE_submission_3250
This submission comes from a modENCODE project of Susan Celniker. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: We will generate over 600 RNA samples in biological triplicate and use them to generate expression profile maps detailing the sites of transcription across the fly genome using whole genome tiling arrays at 38 bp resolution as a broad survey of the transcriptome and 7 bp arrays resolution to identify at high resolution transcripts ends, splice sites, and small RNAs.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: Transcript tiling array analysis. BIOLOGICAL SOURCE: Strain: Y cn bw sp; Developmental Stage: Embryo 4-6h; Genotype: y[1] oc[R3.2]; Gr22b[1] Gr22d[1] cn[1] CG33964[R4.2] bw[1] sp[1]; LysC[1] lab[R4.2] MstProx[1] GstD5[1] Rh6[1];  NUMBER OF REPLICATES: 3;  EXPERIMENTAL FACTORS: Strain Y cn bw sp; Developmental Stage Embryo 4-6h

first: Tom

last: Gingeras

first: Chris

last: Zaleski


title: siRNAs induced long-term liver ApoB mRNA knockdown significantly lowers serum cholesterol in a mouse model with human-like serum lipids

status: Public on Nov 22 2010

city: Rahway

first: Oscar

last: Puig

state: NJ

address: 126 East Lincoln Ave

department: Molecular Profiling Research Informatics

institute: Merck Research Laboratories

summary: Increased serum apoB and associated low density lipoprotein cholesterol (LDL) levels are well correlated with an increased risk of coronary disease.  Apo E-/- and LDLr -/- mice have been extensively used for studies of coronary atherosclerosis.  These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB containing LDL particles.  We describe the development of a new mouse model with a human-like lipid profile.  Ldlr+/- CETP+/- hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation.  To evaluate the apoB pathways in this mouse model, we used novel siRNAs formulated in lipid nanoparticles (LNPs).  ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr+/- CETP+/- mice.   ApoB targeting is specific, dose dependent and shows lipid lowering effects for over three weeks. Although specific TGs were affected by ApoB KD, and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change.  Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.

overall_design: Two oligoes specific for ApoB were used to knock down apoB expression in C57Bl6 or B6-Ldlr(tm1)Tg(APOA1-CETP) mice. Gene expression profiling was performed with Affymetrix Merck Custom Mouse 1.0 microarrays (GPL9734).

first: Marija

last: Tadin-Strapps

first: Lyndon

last: Mitnaul

first: Kenny

last: Wong


title: Gene expression profiling of GPR86 (P2RY13) KO mice

status: Public on Sep 10 2010

summary: The purinergic receptor P2Y13 (P2RY13) has been shown to play a role in the uptake of holo-HDL particles in in vitro hepatocyte experiments. In order to determine the role of P2Y13 in lipoprotein metabolism in vivo, we ablated the expression of this gene in mice and found that P2Y13 knockout mice have lower plasma HDL (17%) and LDL (27%) levels as well as lower fecal concentrations of neutral sterols. In addition, significant decreases were detected in serum levels of fatty acids, glycerol and triglycerides in P2Y13 knockout mice. mRNA profiling analyses showed that ablation of P2Y13 affects hepatic gene expression in a gender-specific manner.  Non-supervised agglomerative cluster analysis showed that expression changes in mice with the same genotype (KO or WT) cluster together and that distinct gene expression clusters can be observed for males and females. Subsequent gene set enrichment analysis showed increased expression of cholesterol and fatty acid biosynthesis genes, while fatty acid beta-oxidation genes were significantly decreased. Liver gene signatures also identified changes in SREBP-regulated and PPARgamma-regulated transcript levels.

overall_design:
Differential gene expression upon P2RY13 gene ablation.
Livers were isolated from male and female wildtype and P2RY13 knockout mice (6 per gender/genotype group, except for 5 per male/WT group). Isolated RNA was subjected to microarray analyses. The results of a one-way ANOVA analysis (p<0.05) showed 3471 transcripts whose relative expression changes were more than 20%.

first: Daniel

last: Blom


title: Dm_y[1]cn[1] bw[1] sp[1]_embryo_4-6h_TotalRNA_p200_224-226-228_7bp

first: DCC

summary:
modENCODE_submission_3248
This submission comes from a modENCODE project of Susan Celniker. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: We will generate over 600 RNA samples in biological triplicate and use them to generate expression profile maps detailing the sites of transcription across the fly genome using whole genome tiling arrays at 38 bp resolution as a broad survey of the transcriptome and 7 bp arrays resolution to identify at high resolution transcripts ends, splice sites, and small RNAs.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf


title: Asymmetric self-renewal associated (ASRA) genes

status: Public on Dec 22 2010

city: Watertown

first: James

last: Sherley

state: MA

address: 64 Grove St.

department: Programs in Regenerative Biology and Cancer Biology

institute: Boston Biomedical Research Institute

summary:
Cell lines geneticially engineered to undergo conditional asymmetric self-renewal were used to identify genes whose expression is asymmetric self-renewal associated (ASRA).  Non-random sister chromatid segregation occurs concordantly with asymmetric self-renewal in these cell lines.
Asymmetric self-renewal occurs when murine embryo fibroblasts that are otherwise p53-null are induced to express physiological levels of wildtype p53 protein (Asym). To distinguish p53-responsive genes that also require induction of asymmetric self renewal (i.e., ASRA genes) and/or non-random sister chromatid segregation for change, an additional control cell line, which continues to symmetrically self-renew (with random sister chromatid segregation) even when p53 is induced, was also compared (Symp53).  This congenic cell line constitutively expresses the type II inosine monophosphate dehydrogenase (IMPDH II; the rate-limiting enzmye for guanine ribonucleotide biosynthesis) and, thereby, prevents p53-induced asymmetric self-renewal and non-random sister chromatid segregation.

overall_design: Three biological replicates of asymmetrically self-renewing cultures (Asym1-3) were compared with cultures that were symmetrically self-renewing - either because they did not express p53 (3 biological replicates, Sym1-3) or they expressed constitutive IMPDH II (i.e., not regulated by p53) as well as p53 (2 biological replicates, Symp53_1 and 2.)

first: Janet

last: Smith

first: Minsoo

last: Noh


title: CVMMS126R_8WG16_N2_L4

status: Public on Jul 07 2010

summary:
modENCODE_submission_599
This submission comes from a modENCODE project of Jason Lieb. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: The focus of our analysis will be elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus. 126 strategically selected targets include key histone modifications, histone variants, RNA polymerase II isoforms, dosage-compensation proteins, centromere components, homolog-pairing facilitators, recombination markers, and nuclear-envelope constituents.
We will integrate information generated with existing knowledge on the biology of the targets, perform ChIP-chip analysis on mutant and RNAi extracts lacking selected target proteins, use extrachromosomal arrays to assess the ability of candidate identified sequence motifs to recruit targets in vivo, identify tissue-specific patterns of selected targets, and create integrated, quantitative models of transcription and whole-chromosome functions.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Keywords: CHIP-chip

overall_design: EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Strain: N2; Developmental Stage: L4 Larva; Genotype: wild type; Sex: mixed Male and Hermaphrodite population;  NUMBER OF REPLICATES: 3;  EXPERIMENTAL FACTORS: Strain N2; temperature 20; Antibody Ab:8WG16:JL:CVMMS126R (target is RNA polII CTD domain unphosophorylated); Developmental Stage L4 Larva

first: Sevinc

last: Ercan

first: Jason

last: Lieb


title: Plant virus detection microarray

status: Public on Jun 30 2010

city: Beijing

first: Yongjiang

last: Zhang

country: China

institute: Chinese Academy of Inspection and Quarantine

summary: 70mer probes were designed to detect plant viruses infection in genus level. This microarray platform is able to detect 169 plant virus species of 13 virus genera.

sample_organism:
Southern bean mosaic virus
Potato virus X
Plum pox virus
Tobacco mosaic virus
Bean pod mottle virus
Arabis mosaic virus
Tomato ringspot virus
Tobacco ringspot virus
unidentified
Zinnia elegans
Prunus necrotic ringspot virus
Taraxacum officinale
Orthotospovirus tomatomaculae

platform_organism: Sobemovirus Carlavirus Capillovirus Potexvirus Potyvirus Tobamovirus Comovirus Nepovirus Tobravirus Cucumovirus Ilarvirus Pomovirus Orthotospovirus

overall_design: Virus sampels were extracted from infected plant hosts. Genomic RNA was extracted and hybridized to the microarray.

first: Jun

last: Yin

first: Shuifang

last: Zhu


title: Cl-amidine treatment reduces autoantibody response in collagen-induced arthritis

status: Public on Aug 21 2010

first: William

last: Robinson

address: 269 Campus Dr

institute: Stanford University

summary: Cl-amidine treatment reduces autoantibody responses in CIA.  Sera were collected from Cl-amidine and control treated mice, and autoantibodies in these samples were profiled using Synovial Antigen Arrays. Significance Analysis of Microarrays (SAM) was used to analyze the antigen array datasets, and identified 8 antigens with a significant difference in autoantibody reactivity (false discovery rate (q) < 5%). Hierarchical clustering was then performed to elucidate the relationships between the autoantibody profiles. The Cl-amidine treated mice clustered together and exhibited lower autoantibody titers against all of the 8 antigens identified by SAM, including autoantibody reactivity to native epitopes derived from cartilage as well as citrulline-modified filaggrin peptides.

platform_organism:
Homo sapiens
Mus musculus

overall_design: Custom-spotted protein arrays were probed with sera samples from individual mice. Eight arrays were probed with sera derived from CIA mice treated with Cl-amidine and five arrays were probed with sera derived from CIA vehicle control mice.

first: Van

last: Willis

first: Alison

last: Gizinski

first: Nirmal

last: Banda

first: Corey

last: Causey

first: Bryan

last: Knuckley

first: Kristen

last: Cordova

first: Yuan

last: Luo

first: Brandt

last: Levitt

first: Magdalena

last: Glogowska

first: Piyanka

last: Chandra

first: Liudmila

last: Kulik

middle: H

last: Arend

first: Paul

last: Thompson

first: V

last: Holers


title: Escherichia coli cells: sorted vs. non-sorted

status: Public on Jul 13 2010

city: Ann Arbor

first: Dongjuan

last: Dai

address: 109 Observatory St

department: Environmental Health Sciences

institute: University of Michigan

summary:
Transcription profile of sorted Escherichia coli cells was compared to that of non-sorted cells to evaluate the effect of sorting process on transcriptome of E. coli. E. coli cells were harvest from planktonic cultures in annular reactor and stored in RNAlater.
Sorting includes 2-min homogenization with OMNI TH homogenizer on ice for E. coli cells pre-stored in RNAlater and then resuspended in nuclease free phosphate buffered saline for sorting with one-step immuno-magnetic separation with anti-E. coli antibody and microbeads on a MACS separator (Miltenyi, Auburn, CA).

overall_design: Two condition experiments: sorted vs non-sorted. Two biological replicates with individually grown and harvested E. coli cells. Each biological replicate has two technical replicates of labeling and hybridization on microarray slides. Each slide has three built-in replicates for each probe.

first: Chuanwu

last: Xi


title: Desai PolII-4H8

first: DCC

last: modENCODE

institute: Ontario Institute for Cancer Research

summary:
modENCODE_submission_174
This submission comes from a modENCODE project of Jason Lieb. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: The focus of our analysis will be elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus. 126 strategically selected targets include key histone modifications, histone variants, RNA polymerase II isoforms, dosage-compensation proteins, centromere components, homolog-pairing facilitators, recombination markers, and nuclear-envelope constituents.
We will integrate information generated with existing knowledge on the biology of the targets, perform ChIP-chip analysis on mutant and RNAi extracts lacking selected target proteins, use extrachromosomal arrays to assess the ability of candidate identified sequence motifs to recruit targets in vivo, identify tissue-specific patterns of selected targets, and create integrated, quantitative models of transcription and whole-chromosome functions.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Keywords: CHIP-chip

overall_design: EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Strain: N2; Developmental Stage: Mixed Embryo; Genotype: wild type; Sex: mixed Male and Hermaphrodite population;  NUMBER OF REPLICATES: 2;  EXPERIMENTAL FACTORS: Strain N2; temperature 20; Antibody ab5408 (target is RNA polymerase II CTD repeat YSPTSPS); Developmental Stage Mixed Embryo

first: Reto

last: Gassmann

first: Arshad

last: Desai


title: ZBTB16, a glucocorticoid response gene in acute lymphoblastic leukemia, interferes with glucocorticoid-induced apoptosis

status: Public on May 07 2010

city: Bolzano

first: Johannes

last: Rainer

address: Via A. Volta 21

department: Institute for Biomedicine

institute: Eurac Researc

summary: Glucocorticoids (GCs) cause apoptosis in lymphoid lineage cells and are therefore widely used in the therapy of lymphoid malignancies. The molecular mechanisms of the anti-leukemic GC effects are, however, poorly understood. We have previously defined a list of GC-regulated candidate genes by Affymetrix-based whole genome comparative expression profiling in children with acute lymphoblastic leukemia (ALL) during systemic GC monotherapy and in experimental systems of GC-induced apoptosis. ZBTB16, a Zink finger and BOZ-domain containing transcriptional repressor, was one of the most promising candidates derived from this screen. To investigate its possible role in GC-induced apoptosis and cell cycle arrest, we performed conditional over-expression experiments in CCRF-CEM childhood ALL cells. Transgenic ZBTB16 alone had no detectable effect on survival, however, it reduced sensitivity to GC-induced apoptosis. This protective effect was not seen when apoptosis was induced by antibodies against Fas/CD95 or 3 different chemotherapeutics. To address the molecular mechanism underlying this protective effect, we performed whole genome expression profiling in cells with conditional ZBTB16 expression. Surprisingly, ZBTB16 induction did not significantly alter the expression profile, however, it interfered with the regulation of several GC response genes. One of them, BCL2L11/Bim, has previously been shown to be responsible for cell death induction in CCRF-CEM cells. Thus, ZBTB16´s protective effect can be attributed to interference with transcriptional regulation of apoptotic genes, at least in the investigated model system.

overall_design: To determine ZBTB16 response genes, C7H2-2C8-ZBTB16#19 and #58 cells (expressing ZBTB16 in a doxycycline-dependent manner) were cultured in duplicates in the absence (treatment “none”) or presence of 400ng/ml doxycycline (treatment “Dox”) for 2h, 6h or 24h. Total RNA was prepared and 1.5 µg RNA subjected to expression profiling on Exon 1.0 microarrays (total of 24 arrays). To assess the effect of ZBTB16 on the GC response, the above cell lines were cultured for 24h in the absence (treatment “Dex”) or presence (treatment “DexDox”) of 200ng/ml doxycycline and subsequently exposed to 10-8M dexamethasone for 6h and 24h and expression-profiled as above resulting in a total of 15 arrays (one of the four 6h replicates (0997_001_58pp6S2_E01_MB_110708.CEL) had to be removed from the following analysis because it didn´t pass quality control).

first: Muhammad

last: Wasim

last: Mansha

first: Michela

last: Carlet

first: Christian

last: Ploner

first: Alexander

last: Trockenbacher

first: Reinhard

last: Kofler


title: Real-time PCR array study for apoptosis genes after radiation exposure in mitochondrial mutant and normal human cells

status: Public on Apr 24 2010

city: Jefferson

first: Rohan

last: Kulkarni

state: AR

address: 3900 NCTR Road

department: Div. of Genetic and Reproductive Toxicology

institute: National Center for Toxicological Research

summary: In double-strand DNA damage repair, non-homologous end joining (NHEJ) is more error prone than homologous recombination repair (HRR), indicating that the relative prevalence of NHEJ may lead to more incorrect repair and thus to increases in chromosome damage. If DNA damage is extensive and cells are unable to repair that damage they typically undergo apoptosis. The mechanism(s) by which cells decide to switch from DNA repair to apoptosis is unknown. Since DNA repair and apoptosis are both energy-demanding processes, the answer may involve ATP utilization. We used human mitochondrial mutant cell lines obtained from people with phenotypic manifestations of compromised ATP generation. We hypothesized that these cells may not have adequate capacity for dealing with the additional demands for ATP required for repairing DNA damage after genotoxic exposure, perhaps making the cells more prone to undergo apoptosis instead of initiating repair. This study describes changes in the expression of genes involved in NHEJ or HRR, as well as genes involved in apoptosis, in one normal and two mitochondrial mutant human cell lines following ionizing radiation exposure. Compared to normal cells, both mutant cell lines showed reduced expression of genes involved in NHEJ and HRR. Analysis of expression changes in genes involved in apoptosis revealed marked increases in expression in the mutants compared to normal cells. These results indicate that following ionizing radiation exposure, mitochondrial mutant cells have decreased levels of mRNA expression of DNA repair genes and increased expression levels of genes involved in apoptosis compared to normal cells. This study provides information that might be useful in characterizing energy dependent processes following exposure to stress or genotoxic agents.

overall_design: Two EBV-transformed human mitochondrial mutant cell lines along with an EBV-transformed normal cell line as a control were exposed to 2 and 4 Gy of ionizing radiation. Sham-radiated cells were used as a control. B2M was used as a housekeeping gene. Reference samples are from the normal cell line. Two replicate array plates were used per sample which had 2 wells per sample.

first: Robert

last: Thomas

first: James

last: Tucker


title: A native chromatin purification system for epigenomic profiling in C. elegans

status: Public on Dec 08 2009

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary:
We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays.

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

overall_design: All experiments were done using two channels per chip, comparing DNAs extracted from either salt-extracted or insoluble chromatin to whole nuclear chromatin, whole nuclear chromatin to randomly fragmented genomic DNA, streptavidin-bound biotin-tagged histone-variant-containing chromatin to salt-extracted chromatin, gel-purified mononucleosomes to whole EDTA-extracted soluble chromatin, or streptavidin-bound biotin-tagged histone-variant-containing chromatin to whole EDTA-extracted soluble chromatin to randomly fragmented DNA from embryo nuceli.

first: Steven

first: Siew-Loon

last: Ooi


title: Genomic Subtypes of Breast Cancer Identified by Array Comparative Genomic Hybridization

status: Public on Jun 24 2010

city: Lund

first: Johan

last: Staaf

address: Medicon Village

country: Sweden

institute: SCIBLU - Swegene Centre for Integrative Biology at Lund University

type:
Expression profiling by array
Genome variation profiling by array

summary: Breast cancer is a profoundly heterogeneous disease with respect to biological and clinical behavior. Gene expression profiling has been used to dissect this complexity and stratify tumors into intrinsic gene expression subtypes associated with distinct biology, patient outcome and different genomic alterations. Additionally, breast tumors occurring in individuals with germline BRCA1 or BRCA2 mutations typically fall into distinct subtypes. We applied global DNA copy number and gene expression profiling in 359 breast tumors. All tumors were classified according to intrinsic gene expression subtypes and included cases from genetically predisposed women. The Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm was used to identify significant DNA copy number aberrations and genomic subgroups of breast cancer. We identified 31 genomic regions that were highly amplified in >1% of the 359 breast tumors. Several amplicons were found to co-occur, the 8p12 and 11q13.3 regions being the most frequent combination besides amplicons on the same chromosomal arm. Unsupervised hierarchical clustering with 133 significant GISTIC regions (66 and 67 with DNA copy number gain and loss, respectively) revealed six genomic subtypes, termed: 17q12, basal-complex, luminal-simple, luminal-complex, amplifier and mixed subtype. Four of them had striking similarity to intrinsic gene expression subtypes and showed association to conventional tumor biomarkers and clinical outcome. However, luminal A-classified tumors were distributed in two main genomic subtypes, luminal-simple and luminal-complex, the former group having better prognosis while the latter group included also luminal B and the majority of BRCA2-mutated tumors. The basal-complex subtype displayed extensive genomic homogeneity and harbored the majority of BRCA1-mutated tumors. The 17q12 subtype comprised mostly HER2-amplified and HER2-enriched subtype tumors and had the worst prognosis. The amplifier and mixed subtypes contained tumors from all gene expression subtypes, the former being enriched for 8p12-amplified cases while the mixed subtype included many tumors with predominantly DNA copy number losses and poor prognosis.

overall_design:
Genomic profiling of 359 breast tumors using tiling BAC aCGH. A number of cases were hybridized as replicates or replicate as dye-swaps.
Gene expression profiling of 359 breast tumors using 55K oligonucleotide microarrays.

first: Göran

last: Jönsson

last: Vallon-Christersson

first: Markus

last: Ringner

first: Karolina

last: Holm

first: Cecilia

last: Hegardt

first: Haukur

last: Gunnarsson

first: Rainer

last: Fagerholm

first: Carina

last: Strand

first: Bjarni

last: Agnarsson

first: Outi

last: Kilpivaara

first: Lena

last: Luts

first: Päivi

last: Heikkilä

first: Kristiina

last: Aittomäki

first: Carl

last: Blomqvist

first: Niklas

last: Loman

first: Per

last: Malmström

first: Håkan

last: Olsson

first: Oskar

last: Johannsson

first: Adalgeir

last: Arason

first: Heli

last: Nevanlinna

first: Rosa

last: Barkardottir

first: Ake

last: Borg


title: Evolution of sex-dependent gene expression in three recently diverged species of Drosophila

status: Public on Jan 12 2010

city: college park

first: Carlos Alberto

last: Machado

department: department of biology

institute: University of Maryland

summary: To identify the transcriptome divergence and sex roles underlying it in recently diverged Drosophia pseudoobscura group, we compare the global gene expression of 7-day-old whole body adults of pure species.

sample_organism:
Drosophila persimilis
Drosophila pseudoobscura
Drosophila pseudoobscura bogotana

overall_design: 7-day-old virgin flies were assayed by species and sex.  At least three isofemale inbred lines were used for each species (D. pseudoobscura; D. persimilis; D. pseudoobscura bogotana).All lines of each species were pooled in equal amounts.Two separate labeling reactions per sample were pooled and hybridized to the Agilent single color (Cyanine 3-CTP dye) arrays. Three different replicates were hybridized for each species and sex.

first: Zifeng

last: Jiang

first: Carlos


title: Identification of potential therapeutic targets for Burkholderia cenocepacia by comparative transcriptomics

status: Public on Dec 01 2009

city: Boston

first: Deborah

middle: R.

last: Yoder-Himes

address: 77 Louis Pasteur Ave, 1038 NRB

department: Microbiology and Molecular Genetics

institute: Harvard Medical School

summary:
[1] Transcription profiling of one Burkholderia cenocepacia clinical isolate, J2315, versus a soil isolate, HI2424, in conditions mimicking CF sputum
[2] Transcription profiling of Burkholderia cenocepacia isolates J2315 and HI2424 in media mimicking CF sputum or the soil environment

overall_design:
[1] J2315 vs. HI2424 cells in the same condition.
[2] Two-condition experiment.
Biological replicates: 4 replicates.

first: James

last: Tiedje

first: Kostantinos

middle: T

last: Konstantinidis


title: Strome NA_MES4FLAG_EEMB

city: Toronto

first: DCC

last: modENCODE

address: MaRS Centre, South Tower, 101 College Street, Suite 800

summary:
modENCODE_submission_202
This submission comes from a modENCODE project of Jason Lieb. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: The focus of our analysis will be elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus. 126 strategically selected targets include key histone modifications, histone variants, RNA polymerase II isoforms, dosage-compensation proteins, centromere components, homolog-pairing facilitators, recombination markers, and nuclear-envelope constituents.
We will integrate information generated with existing knowledge on the biology of the targets, perform ChIP-chip analysis on mutant and RNAi extracts lacking selected target proteins, use extrachromosomal arrays to assess the ability of candidate identified sequence motifs to recruit targets in vivo, identify tissue-specific patterns of selected targets, and create integrated, quantitative models of transcription and whole-chromosome functions.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Keywords: CHIP-chip

overall_design: EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Strain: MES4FLAG tagged by Bombard); Developmental Stage: Early Embryo; Genotype: mes-4(bn73); bnIs15[mes-3 promoter::MES-4::GFP::3xFLAG::mes-4 3' region; Sex: mixed Male and Hermaphrodite population; Transgene: mes-3 promoter::MES-4 coding sequence::GFP::3xFLAG::mes-4 3' sequence;  NUMBER OF REPLICATES: 2;  EXPERIMENTAL FACTORS: Strain MES4FLAG tagged by Bombard); temperature 20; Antibody No Antibody Control (target is Not Applicable); Developmental Stage Early Embryo

first: Taryn

last: Phippen

first: Susan

last: Strome


title: Comparison of Labeling Methods for gene expression analysis on the NimbleGen platform

status: Public on Nov 25 2009

city: Emeryville

first: Mario

last: Ouellet

address: 5885 Hollis St., 4th Floor

institute: Joint BioEnergy Institute

summary:
Comparison of ds-cDNA, Indirect and Direct Random Labeling Methods for gene expression analysis on the NimbleGen platform.
Expression profiles from artemisinic acid-producing S. cerevisiae strain EPY330 and non-producing strain EPY338 are compared for each labeling method tested. The labeling methods and their comparison are described in detail in Ouellet et al., BMC Biotech 2009. Strains were described in detail previously in Ro et al. BMC Biotechnol 2008, 8(1):83 [PMID: 18983675]

overall_design: RNA pools from strains EPY330 (sample A) and EPY338 (sample B) were reverse-transcribed and labeled in triplicate with the ds-cDNA, the Indirect and the new Direct Random method and hybridized in parallel on NimbleGen 4-plex arrays.

first: Aindrila

last: Mukhopadhyay

first: Jay

middle: D

last: Keasaling


title: Gene expression profiling of hypertensive and hypotensive mice

first: Oscar

last: Puig

summary: Hypertension is a condition with major cardiovascular and renal complications, affecting nearly a billion patients worldwide. Few validated gene targets are available for pharmacological intervention, so there is a need to identify new biological pathways regulating blood pressure and containing novel targets for treatment. The genetically hypertensive “blood pressure high” (BPH), normotensive  “blood pressure normal” (BPN),  and hypotensive "blood pressure low" (BPL) inbred mouse strains are an ideal system to study differences in gene expression patterns that may represent such biological pathways. We profiled gene expression in liver, heart, kidney, and aorta from BPH, BPN, and BPL mice and determined which biological processes are enriched in observed organ-specific gene signatures. As a result, we identified multiple biological pathways linked to blood pressure phenotype that could serve as a source of candidate genes causal for hypertension. In order to distinguish causal genes from responsive genes in the kidney gene signature we integrated phenotype associated genes into Genetic Bayesian networks, identifying several novel candidate genes causal for hypertension. The integration of data from gene expression profiling and genetics networks is a valuable approach to identify novel potential targets for the pharmacological treatment of hypertension.

overall_design:
Animals:
BPH/2J, BPL/1J and BPN/3J (142 ± 5 mmHg, 69 ± 1.7 mmHg, 94 ± 6 mmHg, SBP respectively) male mice (Jackson Laboratory) were maintained on a 12:12-h light-dark cycle and fed with standard chow ad libitum in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures were in conformance with the National Research Council's Guide for the Care and Use of Laboratory Animals.
Tissue collection:
Mice (12 weeks old) were euthanized with CO2. Liver, heart, and kidney were flash frozen in liquid nitrogen. Aorta from the aortic root to the left renal artery was rinsed with PBS, immersed in 300ul of RNeasy Lysis buffer (Qiagen), and flash frozen in liquid nitrogen.
RNA preparation:
Tissues (~100mg, 5 mice per strain) were collected independently and homogenized in 2ml of Trizol (Invitrogen). After extraction with 0.4 ml of chloroform, RNA was extracted with SV Total RNA extraction kit (Promega) followed by DNase I treatment and purification using the RNeasy Kit  (Qiagen). RNA was assayed for quality (Agilent Bioanalyzer) and yield (Ribogreen). Kidney, heart and liver RNA was amplified and labeled using a custom automated version of the RT/IVT protocol and reagents provided by Affymetrix. Aorta RNA was amplified and labeled using a custom automated version of the NuGEN Ovation WB protocol (NuGEN). Hybridization, labeling and scanning were according to Affymetrix. All samples were processed independently.
Microarray analysis:
Merck/Affymetrix mouse 1.0 custom arrays monitoring 38384 individual transcripts (25846 Entrez genes) were used. Raw intensity was normalized using the RMA algorithm.

first: I-Ming

last: Wang

first: Ping

last: Cheng

first: Pris

last: Zhou

first: Sophie

last: Roy

first: Doris

last: Cully

first: Mette

last: Peters

first: John

last: Thompson

first: Tian-Quan

last: Cai


title: Genome-wide location analysis of Dnmt3a-mediated epigenetic regulation in murine postnatal subventricular zone (SVZ) neural stem cells (NSCs) [NimbleGen]

status: Public on Jun 25 2010

city: Los Angeles

first: Yi

last: Sun

address: 635, Charles E. Young Dr. South

department: Molecular and Medical Pharmacology

institute: UCLA

summary: DNA methylation at proximal promoters facilitates lineage restriction by silencing cell-type specific genes. However, euchromatic DNA methylation frequently occurs in regions outside promoters. The functions of such non-proximal promoter DNA methylation are unclear. Here we show that the de novo DNA methyltransferase Dnmt3a is expressed in postnatal neural stem cells (NSCs) and is required for neurogenesis. Genome-wide analysis of postnatal NSCs indicates that Dnmt3a occupies and methylates intergenic regions and gene bodies flanking proximal promoters of a large cohort of transcriptionally permissive genes, many of which encode regulators of neurogenesis. Surprisingly, Dnmt3a-dependent non-proximal promoter methylation promotes expression of these neurogenic genes by functionally antagonizing Polycomb repression. Thus, non-promoter DNA methylation by Dnmt3a may be utilized for maintaining active chromatin states of genes critical for development.

overall_design: Chromatin extracted from wild-type (WT) or Dnmt3a-null (KO) SVZ NSCs was immunoprecipitated with indicated antibodies and analyzed by NimbleGen 2.1M mouse whole genome tiling microarrays (a 4-array set covering the entired non-repetitive portion of mouse genome). Whole cell extract (WCE) was used as input controls for IP/WCe experiments. For IP/IP experiments, immunoprecipitated DNA from WT and KO NSCs was directly compared on the same microarrays. For identifying Dnmt3a-dependent DNA methylation at a genome-wide scale, a dye-swap design was employed for comparing DNA methylation levels between WT and KO SVZ NSCs.

first: Hao

last: Wu

first: Volkan

last: Coskun

first: Jifang

last: Tao

first: Wei

last: Xie

first: Weihong

last: Ge

first: Kazuaki

last: Yoshikawa

first: En

last: Li

last: Zhang


title: Relative quantification of miRNAs across multiple experiments

status: Public on Oct 27 2009

city: Bergisch Gladbach

first: Ute

last: Bissels

state: NRW

address: Friedrich-Ebert-Str. 68

department: R&D

institute: Miltenyi Biotec GmbH

summary: MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells.

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design:
We analyzed to which extend the universal reference can be used as a tool for the relative quantification of miRNAs across multiple experiments. We compared the results of direct hybridizations i.e. sample vs. sample to those of indirect hybridizations i.e. sample vs. UR. For the direct hybridizations, we hybridized 5µg liver total RNA vs 5 µg brain total RNA (n = 3) and for the indirect hybridization 5 µg liver or brain total RNA vs UR (5 fmol/miRNA) (n = 3). We calculated the so-called re-ratios for the UR experiments by dividing the signal ratios of the liver vs. UR array by the respective brain vs. UR array gaining a liver vs. brain re-ratio.
Each RNA sample was mixed with 5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled. The RNA mix was hybridized in a dual colour approach to microarrays.
The mean ratios of all probes were normalized to the median of the ratios detected for the spiked 18 synthetic RNA oligonucleotides reverse complement to miRControl 3 probes.


title: Absolute quantification of miRNAs

overall_design:
Total liver RNA was mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled by 3’ ligation. Total RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool (n = 6). The synthetic miRNA pool consisted of 2.5 fmol of each of  891 non redundant miRNAs sequences and miRControl 3 sequences.
The array data was normalized by calculating the median of the miRControl 3 present in the liver and UR sample. The miRNA amount was calculated with respect to the corresponding miRNA in the UR.


title: Genome-wide expression profiling revealed peripheral effects of CB1 inverse agonists in improving insulin sensitivity and metabolic parameters

status: Public on Jun 21 2010

first: Olivia

last: Fong

department: Molecular Profiling

institute: Merck & Co.

summary: Inhibition of cannabinoid receptor 1 (CB1) has shown efficacy in reducing body weight and improving metabolic parameters, with the effects correlating with target engagement in the brain. Recently, the peripheral effects of inhibiting the CB1 receptor has been appreciated through studies in diet-induced obese and liver-specific CB1 KO mice. In this report, we systematically investigated gene expression changes in peripheral tissues of DIO mice treated with the CB1 inverse agonist AM251. CB1 receptor inhibition led to down-regulation of genes within the de novo fatty acid and cholesterol synthetic pathways, including SREBP-1 and -2, and their downstream targets in both liver and adipose tissue. In addition, genes involved in fatty acid Beta-oxidation were up-regulated with AM251 treatment, probably through the activation of PPARalpha. In adipose tissue, CB1 receptor inhibition led to the down-regulation of genes in the TNFalpha signal transduction pathway and possibly to the activation of PPARgamma, both of which would result in improved insulin sensitivity.

overall_design: CB1-/- mice were obtained from A. Zimmer (University of Bonn) (Zimmer et al., 1999) and back-crossed onto C57BL/6J genetic background for ten generations by A. Zimmer before homozygous CB1-/- mice were re-derived at Taconic Farms (German Town, NY) onto the C57BL/6N genetic background.  Male CB1-/- mice and control littermates (n=5-7 in each group) at 2 months of age were fed with regular chow (Teklad 7012, 13 % kcal from fat, 3.41 kcal/g, Harlan Laboratories, Indianapolis, IN, USA) or high fat diet (S3282, 59.4% kcal from fat; 24.5% kcal from carbohydrate; 16.2% kcal from protein; 5.29 kcal/g, Bio-Serv, Frenchtown, NJ, USA) for 14 weeks, and were individually caged 1 week before drug treatment. Vehicle (0.5% methylcellulose) or AM251 (10 mg/kg, Sigma, St Louis, MO, USA) were dosed by oral gavage at 5:00 PM daily for 2 days. Body weight was measured at 5:00 PM on day 1 and 2, and at 10:00 AM on day 3 before tissue collection. Food was measured at 5:00 PM on day 1 and at 10:00 AM on day 3 before tissue collection. Food intake is calculated as the difference in food weight at the start minus at the end of the study. Mice were euthanized by CO2 asphyxiation at 10:00 AM following the second dose.

first: Wenqing

last: Zhao

first: Eric

last: Muise

first: Drew

last: Weingarth

first: Su

last: Qian

first: Tung


title: The network of targeting interactions in chromatin

status: Public on Nov 05 2009

city: Amsterdam

first: Bas

last: van Steensel

address: Plesmanlaan 121

department: division of Molecular Biology

country: Netherlands

institute: Netherlands Cancer Institute

type: Genome binding/occupancy profiling by array

summary:
In eukaryotes, many chromatin proteins together regulate gene expression.
Chromatin proteins often direct the genomic binding pattern of other chromatin proteins, for example by recruitment or competition mechanisms.
The network of such targeting interactions in chromatin is complex and still poorly understood. Based on genome-wide binding maps, we constructed a network model of the targeting interactions among a broad set of 43 chromatin components in Drosophila cells. This model predicts many novel functional relationships. For example, we identified mechanisms that determine the distinct genomic binding patterns of the heterochromatin components HP3 and Su(var)3-7. We also discovered a central role for the remodeling factor Brahma in the targeting of several DNA-binding factors.
Furthermore, the interaction network consists of several sub-networks that regulate distinct groups of genes. Our network model provides a global view of the targeting interplay among dozens of chromatin components.
For this study, we used 36 published datasets, representing various chromatin components,  as well as 7 newly generated DamID profiles. Additionally, we generated DamID profiles of 4 proteins and a profile of differential accessibility in Brm knockdown vs. control cells.

overall_design:
DamID experiments for multiple chromatin proteins was performed in Drosophila cell cultures. Samples were hybridized to spotted cDNA arrays. Every experiment was done at least in duplicate with one sample in the reverse dye orientation. Where indicated, one biological sample was hybridized to two arrays (a and b) in the same dye orientation; values from these arrays were first averaged before averaging dye-swapped biological replicates. Samples represent averaged results of replicates.  A metadata file and a value matrix for the individual replicates are linked below as supplementary files.

Change in chromatin accessibility was probed by Dam methylase expression in white vs. brahma RNAi treated Kc167 cells. Material from two test and control experiments was isolated according to DamID procedure and co-hybridized in reverse dye orientations. Sample represents averaged results of replicates. A metadata file and a value matrix for the individual replicates are linked below as supplementary files. An additional processed data file 'GATCfragments_deltaAcc.gff' whose rows represent normalized values averaged per GATC fragment rather than normalized values per array reporter is linked below as a supplementary file.

first: Ulrich

last: Braunschweig

first: Guillaume

middle: J

last: Filion

first: Menzies

last: Chen

first: Joke

middle: G

last: van Bemmel

first: Trey

last: Ideker


title: Sensitivity and linear dynamic range of miRNA detection

overall_design:
The linear dynamic range and sensitivity of the microarray was measured by hybridizing dilution series of a Universal Reference (UR), an equimolar mixture of synthetic miRNAs.
The UR was hybridized with 10,000 to 1 amol of each individual miRNA. Each individual miRNA and 1 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes was fluorescently labelled by 3’ ligation. The RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool.


title: CGH S2-DRSC Cells

status: Public on Jul 29 2009

first: DCC

last: modENCODE

summary:
modENCODE_submission_725
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: Analysis of copy number variation across modENCODE Drosophila cell line by comparative genomic hybridization (CGH).  We observe numerous copy number variations for the different cell lines, with each cell line having a distinct ploidy signature.

Keywords: CGH

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

overall_design: EXPERIMENT TYPE: CGH. BIOLOGICAL SOURCE 1: Strain: w1118; Developmental Stage: Embryo 0-4h; Genotype: w[1118]; Sex: Unknown; BIOLOGICAL SOURCE 2: Cell Line: S2-DRSC; Tissue: embryo-derived cell-line; Sex: Male;  NUMBER OF REPLICATES: 2;  EXPERIMENTAL FACTORS: Cell Line S2-DRSC

first: David

last: MacAlpine

first: Sara

last: Powell

last: Spana


title: Microarray probe design

overall_design:
Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced.
5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of  816 non redundant miRNAs sequences and miRControl 3 sequences.
Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).


title: Absolute quantification of CD34(+)/CD133(-) hematopoietic stem cells

sample_organism:
Homo sapiens
synthetic construct

overall_design:
The RNA extracted from 7 x 10^ 5 to 1 x 10^ 6 CD34(+)/CD133(-) cells of three different donors was analyzed.
1 µg of respective total RNA was mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled by 3’ ligation. Total RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 2.5 fmol of each of  954 non redundant miRNAs sequences and miRControl 3 sequences.
The array data was normalized by calculating the median of the miRControl 3 present in the CD34(+)/CD133(-) and UR sample. The miRNA amount was calculated with respect to the corresponding miRNA in the UR.


title: CGH Follicle OregonR-mod

summary:
modENCODE_submission_723
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: Most terminally differentiated Drosophila tissues are either polyploid or polytene.  Unlike  normal chromosomes, where the entire chromosome must be replicated exactly once, polytene chromosomes are often differentially replicated with many regions underreplicated and some overreplicated. We will characterize five different polytene tissues using comparative genomic hybridization (CGH) to identify differentially replicated regions of each chromosome. These studies will also identify tissue specific amplicons, where the replication mediated amplification of specific loci is essential for up-regulation of mRNA levels encoding proteins critical for development. The differential replication of polytene chromosomes in Drosophila will provide a unique opportunity to understand how developmental cues and chromosomal domains influence replication initiation.

Keywords: CGH

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

overall_design: EXPERIMENT TYPE: CGH. BIOLOGICAL SOURCE 1: Strain: Oregon-R; Developmental Stage: Embryo 0-4h; Sex: Unknown; BIOLOGICAL SOURCE 2: Strain: Oregon-R; Tissue: oocyte associated follicle cell; Sex: Female;  NUMBER OF REPLICATES: 2;  EXPERIMENTAL FACTORS: Tissue oocyte associated follicle cell

first: Terry

last: Orr-Weaver

first: Thomas

last: Eng


title: CGH Salivary Gland - Y cn bw sp

summary:
modENCODE_submission_720
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: Most terminally differentiated Drosophila tissues are either polyploid or polytene.  Unlike  normal chromosomes, where the entire chromosome must be replicated exactly once, polytene chromosomes are often differentially replicated with many regions underreplicated and some overreplicated. We will characterize five different polytene tissues using comparative genomic hybridization (CGH) to identify differentially replicated regions of each chromosome. These studies will also identify tissue specific amplicons, where the replication mediated amplification of specific loci is essential for up-regulation of mRNA levels encoding proteins critical for development. The differential replication of polytene chromosomes in Drosophila will provide a unique opportunity to understand how developmental cues and chromosomal domains influence replication initiation.

Keywords: CGH

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

overall_design: EXPERIMENT TYPE: CGH. BIOLOGICAL SOURCE 1: Strain: Y cn bw sp; Tissue: larval salivary gland; Genotype: y[1] oc[R3.2]; Gr22b[1] Gr22d[1] cn[1] CG33964[R4.2] bw[1] sp[1]; LysC[1] lab[R4.2] MstProx[1] GstD5[1] Rh6[1]; Sex: Unknown; BIOLOGICAL SOURCE 2: Strain: Y cn bw sp; Developmental Stage: Embryo 0-4h; Sex: Unknown;  NUMBER OF REPLICATES: 1;  EXPERIMENTAL FACTORS: Tissue larval salivary gland


title: CGH Salivary Gland - OregonR

summary:
modENCODE_submission_722
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: Most terminally differentiated Drosophila tissues are either polyploid or polytene.  Unlike  normal chromosomes, where the entire chromosome must be replicated exactly once, polytene chromosomes are often differentially replicated with many regions underreplicated and some overreplicated. We will characterize five different polytene tissues using comparative genomic hybridization (CGH) to identify differentially replicated regions of each chromosome. These studies will also identify tissue specific amplicons, where the replication mediated amplification of specific loci is essential for up-regulation of mRNA levels encoding proteins critical for development. The differential replication of polytene chromosomes in Drosophila will provide a unique opportunity to understand how developmental cues and chromosomal domains influence replication initiation.

Keywords: CGH

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

overall_design: EXPERIMENT TYPE: CGH. BIOLOGICAL SOURCE 1: Strain: Oregon-R Orr-Weaver; Developmental Stage: Embryo 0-4h; Genotype: TBA; Sex: Unknown; BIOLOGICAL SOURCE 2: Strain: Oregon-R Orr-Weaver; Tissue: larval salivary gland; Genotype: TBA; Sex: Unknown;  NUMBER OF REPLICATES: 2;  EXPERIMENTAL FACTORS: Tissue larval salivary gland


title: Transcriptional termini of genes on chromosomes 21-22 (RNA mapping)

status: Public on Jul 28 2009

city: Woodbury

first: Chris

last: Zaleski

state: NY

address: 500 Sunnyside Blvd

institute: Cold Spring Harbor Labs

summary:
The purpose of these experiments was to determine the 5' and 3' transcriptional termini of genes on chromosomes 21-22. Towards this end, we mapped the poly A + RNA isolated from 12 normal tissues and four cell lines.

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

overall_design: polyA+ RNAs from 11 tissues (Brain Frontal Lobe, Brain Hippocampus, Brain Hypothalamus, Cerebellum, Ovary, Placenta, Prostate, Testis, Fetal Kidney, Fetal Spleen, Fetal Thymus; all from BD Clontech) and 5 cell lines (GM06990, HeLaS3, HepG2, K562, tert-BJ) using the BD SMARTTM RACE cDNA amplification kit (BD Clontech Cat. No.634914). A single array with no replicates was run for each sample with a pool of ~23-24 RACE reactions per sample.

last: Gingeras


title: Strome SGF3165_FLAG_MES4FLAG_EEMB

status: Public on May 19 2009

summary:
modENCODE_submission_195

This submission comes from a modENCODE project of Jason Lieb. For full list of modENCODE projects, see http://www.genome.gov/26524648

Project Goal: The focus of our analysis will be elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus. 126 strategically selected targets include key histone modifications, histone variants, RNA polymerase II isoforms, dosage-compensation proteins, centromere components, homolog-pairing facilitators, recombination markers, and nuclear-envelope constituents.We will integrate information generated with existing knowledge on the biology of the targets, perform ChIP-chip analysis on mutant and RNAi extracts lacking selected target proteins, use extrachromosomal arrays to assess the ability of candidate identified sequence motifs to recruit targets in vivo, identify tissue-specific patterns of selected targets, and create integrated, quantitative models of transcription and whole-chromosome functions.

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

overall_design:
EXPERIMENT TYPE: ChIP-chip

BIOLOGICAL SOURCE: Strain: MES4FLAG

DEVELOPMENTAL STAGE: embryo

GENOTYPE: mes-4(bn73); bnIs15

TRANSGENE: mes-3 promoter

NUMBER OF REPLICATES: 3, replicate 1 applied to 3 arrays, replicate 2 applied to 3 arrays, replicate 3 applied to 3 arrays
replicate 3 array 0 (sample id: SGF3165_FLAG_MES4FLAG_EEMB_3 extraction2_array0) is dye swap
replicate 3 array 1 (sample id: SGF3165_FLAG_MES4FLAG_EEMB_3 extraction2_array1) is dye swap
replicate 3 array 2 (sample id: SGF3165_FLAG_MES4FLAG_EEMB_3 extraction2_array2) is dye swap

EXPERIMENTAL FACTORS: Antibody SGF3165 FLAG

first: Taryn

last: Phippen

first: Susan

last: Strome


title: Recombinant and plasma-derived Factor VIII products induce distinct splenic cytokine microenvironments in hemophilia A

status: Public on Apr 30 2009

city: Kingston

first: Mohammad

last: Qadura

address: 88 Stuart St.

department: Pathology

institute: Queen's University

summary:
To investigate the gene expression in dendritic cells after treating hemiphilic mice with HBSS, plasma derived or recombinant factor VIII
Gene expression in dendritic cells were measured 24 hours post treatment

overall_design: Hemophilic mice with treated with either (HBSS, rFVIII or pdFVIII) 24 hours post treatment the spleens were isolated and CD11+ DCs were purified. The gene expression in the DCs were then investigated by microarray studies.

last: Lillicrap


title: Kc167 MCM Localization

summary:
modENCODE_submission_675
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: We will precisely identify sequence elements that direct DNA replication by using chromatin immunoprecipitation of known replication initiation complexes.  These experiments will be conducted in multiple cell types and developmental tissues.

Keywords: CHIP-chip

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

overall_design: EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Cell Line: Kc167; Tissue: embryo-derived cell-line; Genotype: se/e; Sex: Female NUMBER OF REPLICATES: 2;  EXPERIMENTAL FACTORS: Antibody Pan MCM2-7 Antibody

first: Heather