title: Rhizobial adaptation to hosts, a new facet in the legume root-nodule symbiosis

status: Public on Mar 07 2010

summary: Certain alpha- and beta-proteobacteria, the rhizobia, are able to infect legume roots, elicit root nodules, and live therein as endosymbiotic, nitrogen-fixing bacteroids. Host recognition and specificity are the results of consecutive programming events in bacteria and host plants in which important signaling molecules, e.g. plant flavonoids and rhizobial lipooligosaccharides, play key roles. Here, we introduce a new aspect of this symbiosis: the adaptive response to hosts. In contrast to host specificity, which determines early steps in bacteria-plant interaction, the adaptation to hosts refers to late events in mature bacteroids where specific genes are transcribed and translated that help the endosymbionts to meet the disparate environmental requirements imposed by the hosts in which they live. This concept was elaborated with Bradyrhizobium japonicum and three different legumes (soybean, cowpea, siratro). We systematically analyzed and compared the transcriptomes as well as the proteomes in bacteroids from root nodules of the three hosts. Transcripts and proteins were thus identified which are induced in only one of the three hosts. We then focused on those determinants that were congruent in the two data sets of host-specific transcripts and proteins, and arrived at 20 for soybean, 7 for siratro, and 4 for cowpea. One conspicuous gene cluster for a predicted ABC-type transporter, differentially expressed in siratro, was deleted. The corresponding mutant had a symbiotic defect on siratro rather than on soybean or cowpea. This result demonstrates the value of the applied approach and corroborates the host-specific adaptation concept.

sample_organism:
Bradyrhizobium japonicum
Glycine max
Vigna unguiculata subsp. unguiculata
Macroptilium atropurpureum

overall_design: B. japonicum transcriptome was determined for the three different host plants

first: Marion

last: Koch

first: Nathanaël

last: Delmotte

first: Hubert

last: Rehrauer

first: Julia

last: Vorholt

first: Gabriella

last: Pessi


title: Expression differences between inversions and their progenitors in Drosophila melanogaster

status: Public on Dec 06 2010

city: Cambridge

first: Lisa

last: Meadows

address: Downing Street

department: Department of Genetics

country: United Kingdom

institute: University of Cambridge

summary:
Inversions have a breakpoint within a "neighbourhood" of testis expressed genes and the other breakpoint megabases away. The gene expression neighbourhood is therefore intact in the progenitor but disrupted in the inversion.

Keywords: Genome variation profiling by array

overall_design: 12 different inversion stocks, 3 or 4 biological replicates for each, including dye swaps.

middle: A

first: Steven

last: Russell


title: Influence of Gross Ovarian Stage on Transcript Profiles in Fathead Minnow (Pimephales promelas) Ovary Tissue

status: Public on May 01 2010

city: Vicksburg

first: Natalia

last: Vinas

state: MS

address: 3909 Halls Ferry Rd

country: USA

institute: Mississippi State University

summary: Interpretation of toxicogenomic experiments conducted with ovary tissue from asynchronous-spawning small fish species are complicated by background variation in the relative abundance and proportion of follicles at different stages within the ovary tissue sample.  This study employed both real-time quantitative PCR and a 15,000 gene oligonucleotide microarray to examine variation in the fathead minnow (Pimephales promelas) ovarian transcriptional profile as a function of quantitative and qualitative differences in ovarian histology.  Multiple lines of evidence supported the conclusion that variation in the transcriptional profile was primarily dependent on the relative abundance of previtellogenic versus vitellogenic follicles in the ovary tissue.  Due to the relatively small proportions of mature ovulated follicles or atretic follicles in the overall follicle population, few putative molecular markers of maturation, ovulation, or atresia could be identified.  However, among the 460 differentially expressed genes identified in the study, targets including HtrA serine peptidase 3 (htra3), tissue inhibitor of metalloproteinase 3 (timp3), aquaporin 8 (aqp8), transgelin 2 like (tagln2), Nedd4 family interacting protein 2 (ndfip2), chemokine ligand 12a (cxcl12a), midkine related growth factor (mdka), and jagged 1b (jag 1b) exhibited responses and functional properties that support them as candidate molecular markers of significant shift in gross ovarian stage.  Overall, results of this study provide insights into background variation in ovary transcript profiles that should aid and enhance the interpretation of toxicogenomic data generated in experiments conducted with small, asynchronous spawning fish species.

overall_design: Relative abundance of approximately 15,000 RNA transcripts in 26 ovary samples and 4 expelled oocyte (egg) samples, representing the five different histoclasses defined for the present study, was evaluated using fathead minnow oligonucleotide microarrays.  Fathead minnow 15,000 gene arrays were purchased from Agilent (Palo Alto, CA, USA). The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies, Palo Alto, CA) was used for microarray hybridizations following the manufacturer’s protocol and recommendations. One ug of total RNA was used for all hybridizations. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturer’s kits and protocols (Quick Amp Labeling kit; Agilent, Palo Alto, CA). An Axon GenePix® 4000B Microarray Scanner (Molecular Devices Inc., city, state) was used to scan microarray images at 5 μm resolution.

first: Daniel

middle: L

last: Villeneuve

last: Garcia-Reyero

first: Dalma

last: Martinovic

first: Jenna

middle: E

last: Cavallin

first: Nathaniel

middle: D

last: Mueller

first: Leah

middle: C

last: Wehmas

first: Michael

last: Kahl

first: Anne

last: Linnum

first: Edward

middle: J

last: Perkins

first: Gerald

middle: T

last: Ankley


title: A high-resolution transcriptome map of Ectocarpus siliculosus

status: Public on Apr 07 2010

city: Los Altos

first: Manoj

last: Samanta

state: CA

address: 2240 Homestead Court #309

institute: Systemix Institute

type: Expression profiling by genome tiling array

summary:
Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. The brown algae are also important because they are one of only a very small number of eukaryotic lineages that have evolved complex multicellularity. This work used whole genome tiling array approach to generate a comprehensive transcriptome map of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for the brown algae.
Keywords: high-resolution tiling array, brown algae, ectocarpus

overall_design: The slides were hybridised with two, labelled samples: 1) a mixture of labelled cDNA corresponding to RNA samples from mature sporophytes and gametophytes and from immature sporophytes stressed either in high salt medium or by addition of hydrogen peroxide and 2) genomic DNA as a control.

middle: P

first: Delphine

last: Scornet

first: J

middle: M

last: Cock


title: The PhyR-sigmaEcfG signaling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum

status: Public on Jul 01 2009

city: Zürich

first: Hans-Martin

last: Fischer

address: Vladimir-Prelog-Weg 4

department: Microbiology

country: Switzerland

institute: ETH Zürich

summary: PhyR is an unusual type of response regulator consisting of a receiver domain and an extracytoplasmic function (ECF) sigma factor-like domain. It was recently described as a master regulator of general stress response in Methylobacterium extorquens. Orthologues of this regulator are present in essentially all free-living Alphaproteobacteria. In most of them, phyR is genetically closely linked to a gene encoding an ECF sigma factor. Here, we investigate the role of these two regulators in the soybean symbiont Bradyrhizobium japonicum USDA110. Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF sigma factor sigma(EcfG) are involved in heat shock and desiccation resistance upon carbon starvation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Microarray analysis revealed that PhyR and sigma(EcfG) control congruent regulons suggesting both regulators are part of the same signalling cascade. This finding was further substantiated by in vitro protein-protein interaction studies which are in line with a partner-switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. The large number of genes of unknown function present in the PhyR/sigma(EcfG) regulon and the conspicuous symbiotic phenotype suggest that these regulators are involved in the Bradyrhizobium-legume interaction via yet undisclosed mechanisms.

overall_design: Comparative analysis of the B. japonicum phyR mutant 8402, ecfG mutant 8404 mutant and the wild type during exponential growth in rich medium (PSY) and after 24 hours starvation in carbon source-free minimal medium

first: B

last: Gourion

first: S

last: Sulser

last: Frunzke

first: A

last: Francez-Charlot

first: P

last: Stiefel

first: G

last: Pessi

last: Vorholt

first: H


title: Drosophila MSL complex globally acetylates H4 Lys16 on the male X chromosome for dosage compensation

status: Public on Jun 10 2009

city: Boston

first: Peter

last: Park

state: MA

address: 10 Shattuck St

department: Center for Biomedical Informatics

institute: Harvard Medical School

type: Genome binding/occupancy profiling by genome tiling array

summary: Drosophila MSL complex binds the single male X chromosome to upregulate gene expression to equal that from the two female X chromosomes.  However, it has been puzzling that ~25% of transcribed genes on the X do not stably recruit MSL complex.  Here, we find that almost all active genes on the X are associated with robust H4 Lys16 acetylation (H4K16ac), the histone modification catalyzed by MSL complex.  The distribution of H4K16ac is much broader than that of MSL complex, and our results favor the idea that chromosome-wide H4K16ac reflects transient association of MSL complex, occurring through spreading or chromosomal looping.  Our results parallel those of localized Polycomb repressive complex and its more broadly distributed H3K27me3 chromatin mark, suggesting a common principle for the establishment of active and silenced chromatin domains

overall_design: For ChIP-on-chip experiments in larvae, 1000 third instar larvae were collected and chromatin was prepared as described previously. Five IPs were performed for each of the two biological replicates. For SL2 cells and male larvae, one ChIP-on-chip experiment was performed using anti-H4K16ac from Serotec (AHP417) (1-2 ul/IP). However, this antibody was discontinued, and an additional experiment for each was performed using anti-H4K16ac from Upstate/Millipore (07-329) (3 ul/IP). Both replicates for Kc cells and female larvae were also performed using anti-H4K16ac from Upstate/Millipore. Replicate experiments were highly reproducible.

first: Marnie

last: Gelbart

first: Erica

last: Larschan

first: Shouyong

last: Peng

first: Mitzi

middle: I

last: Kuroda


title: LCM-based microarray analysis of neuronal vulnerability in the mouse model of Sanfilippo syndrome (MPSIIIB)

status: Public on Apr 22 2009

city: Torrance

first: Stanislav

last: Karsten

address: 1124 Carson Street

department: Neurology

institute: Harbor-UCLA

summary: Sanfilippo syndrome type B (MPS III B) is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of N-acetylglucosaminidase and lysosomal accumulation of heparan sulfate.  In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate,   but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances.  We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu -/- and Naglu +/- mice.  Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls.  The highest increase in gene expression (6- to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs, which encodes lysozyme, but  accumulation of lysozyme protein was seen in MEC neurons only.   Because of a report that lysozyme induced the formation of hyperphosphorylated tau (P-tau) in cultured neurons, we searched for P-tau by immunohistochemistry.  P-tau was found in MEC of Naglu -/- mice, in the same neurons as lysozyme.  In older mutant mice, it was also seen in the dentate gyrus, an area important for memory.  Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments - P-tau aggregates characteristic of tauopathies, a group of age-related dementias that includes Alzheimer disease.  Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy.

overall_design: Two-condition experiment, Naglu-/- (affected) vs. Naglu+/- (unaffected control) of neurons from two adjacent brain regions: medial entorhinal cortex (MEC) and lateral entorhinal cortex (LEC). Biological replicates: 3 MEC Naglu-/-, 3 MEC Naglu+/-, 3 LEC Naglu-/- and 3 LEC Naglu+/-. Comparisons were made between 3 pairs of mutant and control female littermates for MEC and LEC neurons with dye switch duplicates, for a total of 12 microarrays.

first: Lili

last: Kudo

first: Kazuhiro

last: Ohmi


title: Expression data from porcelain crabs following heat or cold stress

status: Public on Sep 21 2009

city: Tiburon

first: Jonathon

middle: H

last: Stillman

address: 3152 Paradise Drive

department: Romberg Tiburon Center

institute: San Francisco State University

summary:
Porcelain crabs, Petrolisthes cinctipes, live in the marine intertidal zone and routinely experience thermal stress.  Genes involved in heat shock responses are generally upregulated following heat stress, differentially expressed genes are involved in different cellular functions.
We used microarrays to detail the global programme of gene expression underlying responses to thermal stress and identified distinct classes of up-regulated and down-regulated genes during this process.

overall_design: Crabs were collected from the field and returned to the laboratory where they were given a heat or cold stress or held under control conditions for the period of time during the thermal stress.

first: Abderrahmane

last: Tagmount


title: Depolymerization of plant cell wall glycans by symbiotic human gut bacteria (Bacteroides ovatus)

status: Public on May 01 2011

city: Ann Arbor

first: Eric

middle: Charles

last: Martens

state: MI

address: 1150 West Medical Center Drive

department: Dept. of Microbiology and Immunology

institute: University of Michigan Medical School

summary: Symbiotic bacteria inhabiting the distal human gut have evolved under intense pressure to utilize complex carbohydrates, predominantly plant cell wall glycans abundant in our diets. These substrates are recalcitrant to depolymerization by digestive enzymes encoded in the human genome, but are efficiently targeted by some of the ~103-104 bacterial species that inhabit this niche. These species augment our comparatively narrow carbohydrate digestive capacity by unlocking otherwise unusable sugars and fermenting them into host-absorbable forms, such as short-chain fatty acids. We used phenotype profiling, whole-genome transcriptional analysis and molecular genetic approaches to investigate complex glycan utilization by two fully sequenced and closely related human gut symbionts: Bacteroides thetaiotaomicron and Bacteroides ovatus. Together these species target all of the common glycosidic linkages found in the plant cell wall, as well as host polysaccharides, but each species exhibits a unique ‘glycan niche’: in vitro B. thetaiotaomicron targets plant cell wall pectins in addition to linkages contained in host N- and O-glycans; B. ovatus uniquely targets hemicellulosic polysaccharides along with several pectins, but is deficient in host glycan utilization.

platform_organism:
Bacteroides thetaiotaomicron VPI-5482
Bacteroides sp. WH2
Blautia obeum ATCC 29174
[Ruminococcus] torques ATCC 27756
Dorea longicatena DSM 13814
[Clostridium] scindens ATCC 35704
Bacteroides ovatus ATCC 8483
Bacteroides uniformis ATCC 8492
Faecalibacterium prausnitzii M21/2
Bacteroides caccae ATCC 43185
Collinsella aerofaciens ATCC 25986
Thomasclavelia spiroformis DSM 1552
Phocaeicola vulgatus ATCC 8482
Parabacteroides distasonis ATCC 8503
Agathobacter rectalis ATCC 33656

overall_design: Bacteroides ovatus bacteria were grown either in vitro on defined complex glycan sources, or in vivo in the intestinal tract of gnotobiotic mice fed variable diets. Increased in vitro gene expression was used to indicate the genes required for metabolism of complex glycans and compared to in vivo transcriptional activity to determine expression in the mouse gut.

first: Jeffrey

last: Gordon


title: microarray summer mortality insitu oyster

status: Public on Aug 15 2009

city: Plouzane

first: Arnaud

last: Huvet

address: Ifremer centre de Brest BP 70

department: Physiologie Fonctionnelle des Organismes Marins

country: France

institute: IFREMER

summary: Summer mortality of the Pacific oyster Crassostrea gigas is the result of a complex interaction between oysters, their environment and pathogens. Heredity appears to be a major factor determining the sensitivity of oysters to summer mortality, allowing resistant (R) and susceptible (S) lines to be produced. We conducted genome-wide expression profiling of R and S gonads during the 3-month period preceding a summer mortality event using a 9K cDNA microarray that we designed. This transcriptional analysis provides new indications to define markers for Quantitative Trait Loci searches and functional studies, and evaluates the potential role of each gene in the resistance to summer mortality

overall_design:
For microarray analysis, R and S oysters were sampled four times (dates 1 to 4: May 9, May 25, June 6, and June 20, respectively). On each date, 3 replicates of 8 oysters were sampled from each line (R and S) and the gonads prepared for total RNA extraction. Furthermore, the entire tissues of 10 wild oysters were collected, pooled and homogenized to constitute a single total RNA sample for use as a reference in all slide hybridizations and RT-PCR analysis.

For microarray hybridizations, 5µg of total RNA were directly labeled by reverse transcription and then purified using the Direct ShipShot Labeling kit (Promega). This reaction was performed for each of the 24 gonad samples, with Cy5 (red) incorporation. The reference sample was Cy3-labeled (green) in 24 separate tubes following the same protocol. The 24 Cy3-labeled cDNAs were next pooled, and then divided once more into 24 samples to obtain a homogeneous reference.

Equimolar amounts of cDNA samples and cDNA reference labeled with Cy5 and Cy3, respectively, were SpeedVac evaporated and mixed into a single pool with the hybridization buffer (ChipHyb™ hybridization buffer, Ventana Discovery, Tucson, AZ, USA). They were then co-hybridized on the same microarray slide, in a Ventana hybridization station (Ventana Discovery, Tucson, AZ, USA).

The data submitted here correspond to the mean of the three replicates for each line and each date, representing 8 samples :
gonad_resistant_date1
gonad_sensitive_date1
gonad_resistant_date2
gonad_sensitive_date2
gonad_resistant_date3
gonad_sensitive_date3
gonad_resistant_date4
gonad_sensitive_date4

first: Elodie

last: Fleury

first: Jeanne

last: Moal

first: Viviane

last: Boulo

first: JeanYves

last: Daniel

first: David

last: Mazurais

first: Alain

last: Hénaut

first: Charlotte

last: Corporeau

first: Pierre

last: Boudry

first: Pascal

last: Favrel


title: Dynamic histone H3 epigenome marking during the intraerythrocytic cycle of P. falciparum

status: Public on May 18 2009

city: Nijmegen

last: Stunnenberg

address: Geert Grooteplein 28

department: Department of Molecular Biology (274)

institute: Radboud University

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

overall_design: Refer to individual Series


title: Repertoire and evolution of miRNA genes in four divergent nematode species

status: Public on Sep 08 2009

city: Groningen

first: Eugene

last: Berezikov

address: Antonius Deusinglaan 1

department: European Research Institute for the Biology of Ageing

country: Netherlands

institute: University Medical Center Groningen

type: Non-coding RNA profiling by high throughput sequencing

summary: We have performed small RNA sequencing in the nematodes Caenorhabditis elegans, C. briggsae, C. remanei and Pristionchus pacificus, which have diverged up to  400 million years ago, to establish the repertoire and evolutionary dynamics of miRNAs in these species. In addition to previously known miRNA genes from C. elegans and C. briggsae we demonstrate expression of many of their homologs in C. remanei and P. pacificus, and identified in total more than 100 novel expressed miRNA genes, the majority of which belong to P. pacificus. More than half of all identified miRNA genes were found to be conserved at the seed level in all four nematode species, whereas only a few miRNAs appear to be species-specific. In our compendium of miRNAs we observed evidence for known mechanisms of miRNA evolution, including antisense transcription and arm switching, as well as miRNA family expansion through gene duplication. In addition, we identified a novel mode of miRNA evolution, termed ‘hairpin shifting’, in which an alternative hairpin is formed with up- or downstream sequences, leading to shifting of the hairpin and creation of novel miRNA* species. Finally, we identified 21U-RNAs in all four nematodes, including P. pacificus, where the upstream 21U-RNA motif is more diverged. However, the genomic distribution of 21U-RNA clusters in P. pacificus appears more scattered throughout the genome as compared to C. elegans. The identification and systematic analysis of small RNA repertoire in four nematode species described here provides a valuable resource for understanding the evolutionary dynamics of miRNA-mediated gene regulation.

sample_organism:
Caenorhabditis briggsae
Caenorhabditis elegans
Caenorhabditis remanei
Pristionchus pacificus

overall_design: Small RNAs were cloned from mixed stage animals. Sequencing was performed using the 454 GS FLX platform.


title: Mapping of H3, H3K4me3 and H3K9ac on Ring and Schizont stages of P. falciparum

summary:
Epigenome profiling has led to the paradigm that promoters of active genes are decorated with H3K4me3 and H3K9ac marks.
These data on synchronized parasites reveals significant developmental stage specificity of the epigenome. In rings, H3K4me3 and H3K9ac are homogenous across the genes marking active and inactive genes equally, whilst in schizonts they are enriched at the 5’end of active genes.

overall_design: Chip-chip (and cDNA) from Plasmodium falciparum strain NF54 Rings and Schizonts with H3, H3K4me3 and H3K9ac.

first: A

last: Salcedo-Amaya

first: M

last: van Driel

first: B

last: Alako

middle: B

last: Trelle

last: van den Elzen

last: Cohen

first: E

last: Janssen-Megens

last: Vegte-Bolmer

first: R

middle: R

last: Selzer

last: Iniguez

last: Green

last: Sauerwein

first: O

middle: N

last: Jensen

first: H

middle: G


title: Differential IFNa-stimulated gene expression profiles in PLZF-inducible U937T monocyte cells

status: Public on May 22 2009

city: Bethesda

first: Michelle

last: Holko

state: MD

address: 45 Center Dr

department: NCBI

institute: GEO

summary: Sensitivity to Interferon (IFN) is determined by a complex coordination of genetic and environmental factors. A previous experiment using two renal cancer cell lines differing markedly in their response to IFN were analyzed for their ISG profiles in order to determine gene expression changes associated with IFN sensitivity (Holko M, Williams BR. Functional annotation of IFN-alpha-stimulated gene expression profiles from sensitive and resistant renal cell carcinoma cell lines. J Interferon Cytokine Res 2006 Aug;26(8):534-47). Higher and more persistent expression of a subset of ISGs was noted in the IFN-sensitive RCC1 cells when compared with the relatively IFN-insensitive ACHN cells. A subset of Interferon stimulated genes (ISGs) whose sustained expression correlates with heightened IFN sensitivity were analyzed for functional and sequence similarities. This subset predominately contains genes involved in transcription, and most were found to contain in their promoters binding sites for promyleocytic zinc finger factor (PLZF), a transcriptional repressor identified by virtue of its role in the etiology of Acute Promyelocytic Leukemia (APL).  Analysis of gene expression in a lymphoid cell line with inducible PLZF expression reveals that increased expression of immune system related genes including ISGs, depends on PLZF expression.  Chromatin Immunoprecipitation assays show direct association between PLZF and in silico identified PLZF binding sites in ISG promoters.  This study reveals a novel interaction between PLZF and IFN signaling.

overall_design: A time course of IFNa treatment (0, 6, 16, 24 hrs) was performed in tetetracycline induced PLZF overexpressing and control U937T:PLZF45 cells.  For the 0 hr time point, PLZF overexpression (Cy5) was compared with control cells (Cy3).  For the 6, 16, and 24 hr IFN treated time points, treated RNA was labelled with Cy5 and non-IFN treated RNA with Cy3.  Each IFN treatment time point was performed on control and PLZF-overexpressing cells respectively.

first: Melanie

last: McConnell

first: Windy

last: Berkofsky-Fessler

first: Jonathan

last: Licht

first: Bryan

last: Williams


title: Mapping of H3, H3K4me3, H3K9me3, and H3K9ac on mixed asexual stages of P. falciparum

last: Stunnenberg

summary:
Epigenome profiling has led to the paradigm that promoters of active genes are decorated with H3K4me3 and H3K9ac marks.
Data revealed an extensively euchromatic epigenome with heterochromatin restricted to variant surface antigen gene families (VSA) and a number of genes hitherto unlinked to VSA. The vast majority of the genome shows an unexpected pattern of enrichment of H3K4me3 and H3K9ac at intergenic regions and depletion at genes.

overall_design: Chip-chip (and cDNA) from Plasmodium falciparum strain NF54 asexual blood stages with H3, H3K4me3, H3K9me3 and H3K9ac

last: Salcedo-Amaya

last: van Driel


title: The effects of LH ablation/replacement versus steroid ablation/replacement on gene expression in primate corpora lutea

status: Public on Jan 26 2009

city: Beaverton

first: Cecily

middle: Vauna

last: Bishop

state: OR

address: 505 NW 185th Ave

department: Division of Reproductive & Developmental Sciences

institute: Oregon National Primate Research Center

summary:
This study was designed to provide a genome-wide analysis of the effects of luteinizing hormone (LH) ablation/replacement versus steroid ablation/replacement on gene expression in the developed corpus luteum (CL) in primates during the menstrual cycle. Naturally cycling, female rhesus monkeys were left untreated (Control; n = 4) or received one of the following treatments for three days beginning on Day 9 of the luteal phase: daily injection of the gonadotropin-releasing hormone (GnRH) antagonist (Antide; n = 5), Antide + recombinant human LH (A+LH; n = 4), Antide + LH + the 3b-HSD antagonist Trilostane (A+LH+TRL; n = 4), and Antide + LH + TRL + progesterone replacement with a synthetic progestin R5020 (A+LH+TRL+ R5020; n = 5). On Day 12 of the luteal phase, CL were removed and samples of RNA from individual CL were fluorescently labeled and hybridized to Affymetrix™ rhesus macaque total genome microarrays. The greatest number of altered transcripts was associated with the ablation/replacement of LH, while ablation/replacement of progestin affected fewer transcripts. Replacement of LH during Antide treatment restored expression of most transcripts to control levels. Real-time PCR validation of a subset of transcripts revealed that most expression patterns were similar between microarray and real-time PCR. Analysis of protein levels were subsequently determined for 2 of the transcripts differentially expressed by real-time PCR. This is the first genome-wide analysis of LH and steroid regulation of gene transcription in the developed primate CL. Further analysis of novel transcripts identified in this data set can clarify the relative role for LH and steroids in CL maintenance and luteolysis.

Keywords: LH/steroid ablation/replacement in primate mid-late luteal phase corpora lutea

overall_design: 22 samples from Rhesus Macaque corpus luteum hybridized to individual Rhesus Affymentrix Gene Chip Arrays. 5 treatment groups, with at least 4 replicates per treatment.

middle: V

first: Jon

last: Hennebold

first: Richard

last: Stouffer


title: Ago-1 and TRIM32-bound small RNAs from E14.5 mouse brain.

status: Public on Mar 10 2009

last: Berezikov

summary: In the mouse neocortex, neural progenitor cells generate neurons through repeated rounds of asymmetric cell division. How distinct fates are established in their daughter cells is unclear. We show here that the TRIM-NHL protein TRIM32 segregates asymmetrically during progenitor division and induces neuronal differentiation in one of the two daughter cells. TRIM32 is highly expressed in differentiating neurons. In both horizontally and vertically dividing progenitor cells, TRIM32 distribution becomes polarized in mitosis so that the protein is enriched in one of the two daughter cells. While TRIM32 overexpression induces cell cycle exit and neuronal differentiation, TRIM32 RNAi causes both daughter cells to proliferate and prevents the initiation of a neuronal differentiation program . TRIM32 ubiquitinates and degrades the transcription factor c-Myc but also binds Argonaute-1 and thereby increases the activity of specific micro-RNAs. We show that Let-7 is one of the TRIM32 targets and is required and sufficient for neuronal differentiation. Our data suggest that the asymmetric segregation of a micro RNA regulator controls self renewal in the mammalian brain.

overall_design: small RNA from total mouse brain, Ago-1 and TRIM32 IPs were cloned and sequenced using 454 GS FLX system.


title: Stimulation of Human and Rat Islet β-Cell Proliferation with Retention of Function by Nkx6.1

status: Public on Oct 11 2008

city: Chapel Hill

last: Schisler

state: NC

address: MBRB, Rm 2340C

department: McAllister Heart Institute

institute: The University of North Carolina at Chapel Hill

summary:
The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet β-cell development, but its effects on adult β-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3H] thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-βGAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are β cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [3H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating β-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of β-cell mass in treatment of both major forms of diabetes.

Keywords: Insulin secretion, islet biology, transcription factor, cell cycle regulation, diabetes

overall_design: We utilized a “sample x reference” experimental design strategy in which RNA extracted from rat pancreatic islets was hybridized to the microarray slide in the presence of labeled rat reference RNA (RRR, Stratagene, LaJolla, CA).  Cultures were treated with adenoviruses expressing either the hamster form of Nkx6.1 or the beta-galactosidase enzyme.  5 biological replicates each representing independent islet isolations were used for microarray analysis.  Briefly, five hundred nanograms of total RNA were used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5.  Labeled subject cRNA was co-hybridized to Operon rat 27K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled RRR. Slides were hybridized, washed, and scanned on a Gene Pix 5000 microarray scanner.

first: Patrick

last: Fueger

first: Daniella

middle: A

last: Babu

first: Hans

last: Hohmeier

first: Jeffery

middle: S

last: Tessem

first: Danhong

last: Lu

first: Thomas

last: Becker

first: Bashoo

last: Naziruddin

first: Marlon

last: Levy

first: Raghavendra

last: Mirmira

first: Christopher

last: Newgard


title: H3K4me3 location in U937 cells

status: Public on Aug 29 2008

city: Chicago

first: Elizaveta

last: Benevolenskaya

state: IL

address: 900 S. Ashland Ave.

department: Biochemistry and Molecular Genetics

institute: University of Illinois at Chicago

summary:
H3K4me3 enrichment at proximal promoter regions
Keywords: ChIP-on-chip data

overall_design: We describe that RBP2 shows high correlation with the presence of H3K4me3. Biological replicates: chromatin immunoprecipitations were performed independently in parallel


title: The MSL3 chromodomain directs a key targeting step for dosage compensation of the Drosophila

status: Public on Nov 19 2008

first: Peter

last: Park

summary: Active genes on the X chromosome of Drosophila males are upregulated by the Male-specific lethal (MSL) complex containing five MSL proteins and two non-coding roX RNAs. To probe the targeting mechanism, we have solved the structure of the MSL3 chromodomain, designed point mutations in key residues that disrupt putative methyl-lysine recognition, and tested their effect on full length MSL3 function. Transgenic males expressing these site-directed point mutants or MSL3short, a naturally occurring MSL3 form lacking the chromodomain, are unhealthy and developmentally delayed. Genomewide analyses of the binding patterns of these mutants support a two-step model: the first step is chromodomain-independent association with “chromatin entry sites” carrying GA-rich MSL recognition elements (MREs). The second step involves spreading from entry sites to the majority of active genes on the X. Either deleting or introducing point mutations in the MSL3 chromodomain disrupts this second step. In vitro studies demonstrate that chromodomain mutants have diminished interaction with recombinant nucleosomes methylated at H3K36. We propose that MSL spreading depends, to a large extent, on the integrity of the MSL3 chromodomain to interact with lysine-methylated nucleosomes.

overall_design: Chromatin immunoprecipitation using TAP-tag was performed as described previously (Alekseyenko et al., 2006). The immunoprecipitated material was eluted from the beads by adding 10 μL (100 U) of AcTEV protease into 450 μL of TEV buffer followed by incubation at 25°C for 1 h. To reverse the cross-links, samples were brought to 0.3M NaCl and 1% SDS and incubated at 65°C for 12 h. The samples were then extracted with phenol/chloroform/isoamyl alcohol followed by chloroform, and precipitated by ethanol in the presence of glycogen. The resulting precipitated DNA and the input DNA were amplified using a DNA linker as described (Strutt et al., 1997). LM-PCR (25 cycles) was performed using Platinum Pfu (Invitrogen). The resulting DNA was labeled and hybridized to arrays by NimbleGen Systems, Inc. Two-channel tiling arrays containing 388,000 probes were designed based on FlyBase 3.2. Chromosomes X and 19.6Mb of 2L were covered. The ChIP sample of interest was hybridized on one channel and genomic DNA was used as the reference on the other  channel. Each ChIP-chip experiment was performed in duplicate.

first: Tuba

last: Sural

first: Shouyong

last: Peng

first: Bing

last: Li

first: Jerry

middle: L

last: Workman

first: Mitzi

last: Kuroda


title: Khd1p associated mRNAs

status: Public on Aug 26 2008

city: ETH Hoenggerberg, Zuerich

first: Andre

last: Gerber

address: Wolfgang Pauli Str. 10, HCI H396

department: Institute of Pharmaceutical Science

institute: Swiss Federal Institute of Technology

summary:
RNA-coimmunopurifications with TAP-tagged Khd1 protein from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control (Gerber et al., 2004). Cells were grown to midlog phase in rich media and harvested by centrifugation. TAP-tagged Khd1 was affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input) and from purified protein samples by phenol-chloroform extraction. RNAs were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/ dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004.
An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
Strain Name: yeast strain with/without TAP-tagged Khd1
Keywords: all_pairs

overall_design: Computed


title: A sequence motif within chromatin entry sites directs MSL establishment on the Drosophila X chromosome

status: Public on Aug 22 2008

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Dosage compensation serves as a model for understanding how chromatin-modification enzymes are targeted to initiate and maintain gene regulation. In Drosophila, the MSL complex associates with active genes specifically on the male X chromosome to acetylate histone H4 at lysine 16, and increase expression of most X-linked genes approximately two-fold. To date, no DNA sequence has been discovered to explain the specificity of MSL binding. We previously hypothesized that sequence-specific targeting occurs at initiation or “chromatin entry sites”, but binding to the majority of sites is sequence-independent. Here we characterize more than 150 potential entry sites by ChIP-chip and ChIP-seq and discover a common GA-rich MSL recognition element (MRE). The motif is only slightly enriched on the X chromosome (~2 fold), but this is doubled when considering its preferential location within or 3’ to active X-linked genes (>4 fold enrichment). When inserted on an autosome, a newly identified site can direct local MSL spreading to flanking active genes on the autosome. These results provide strong evidence for both sequence-dependent and -independent steps in MSL targeting of dosage compensation to the male X chromosome.

overall_design:
ChIP-chip: msl3 mutant embryos (1 g) were collected for chromatin preparation as described in Alekseyenko et al. (2006). Immunoprecipitation was performed with anti-MSL2 antibody, as in Larschan et al. (2007). Immunoprecipitated DNA was isolated, amplified and hybridized to NimbleGen tiling arrays as in Alekseyenko et al (2006). We performed two replicates for each type of ChIP-chip experiment.
ChIP-seq: Immunoprecipitated DNA from Clone 8 cells was isolated as in Alekseyenko et al. (2006). DNA amplification was performed using the Illumina Sample Prep Kit with minor modifications. 1 µl adapter oligo mix was used per 100 ng ChIP DNA. The ligation mix was first amplified as detailed in the protocol and then the PCR products (3-8 µg) were gel-fractionated using Biorad Certified Low Range Ultra Agarose. Different fractions (100-200 bp, 200-300 bp, 300-400 bp) were collected. Purified DNA was run on the Solexa Genome Analyzer. A detailed protocol for Solexa sample preparation is available upon request

first: Artyom

last: Alekseyenko

first: Erica

last: Larschan

first: Andrey

last: Gorchakov

first: Ok-Kyung

last: Lee

last: Kharchenko

first: Sean

last: McGrath

first: Charlotte

last: Wang

first: Elaine

last: Mardis


title: RBP2 location analysis

summary:
RBP2 (JARID1A/RBBP2) enrichment at proximal promoter regions in differentiating promonocytic U937 cells and in the osteosarcoma SAOS-2 cells
Keywords: differentiation treatment, ChIP-on-chip analysis

overall_design: We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis. We describe that RBP2 target genes are separated into two functionally distinct classes: differentiation independent and differentiation dependent genes.Three condition experiment, U937 cells treated with 100 nM 12-O-tetradeconoyl-phorbol 13-acetate (TPA) (Sigma) for two different time points (27 hrs and 96 hrs) and untreated (0 hrs time point or no TPA treatment). In addition, processed data for the unrelated SAOS-2 cells are included. Biological replicates: threechromatin immunoprecipitations (ChIP) performed independently in parallel

first: William

last: Kaelin Jr


title: A methodology for the estimation of DNA methylation levels based on microarray derived MeDIP-enrichment

status: Public on Jul 21 2008

city: New Haven

first: Annette

last: Molinaro

state: CT

address: 60 College St

institute: Yale University

type: Methylation profiling by genome tiling array

summary:
DNA methylation is an important component of epigenetic modifications that influences the transcriptional machinery and is aberrant in many human diseases. Several methods have been developed to map DNA methylation for either limited regions or genome-wide. In particular, antibodies specific for methylated CpG have been successfully applied in genome-wide studies. However, despite the relevance of the obtained results, the interpretation of antibody enrichment is not trivial. Of greatest importance, the coupling of antibody-enriched methylated fragments with microarrays generates DNA methylation estimates that are not linearly related to the true methylation level. Here, we present an experimental and analytical methodology to obtain enhanced estimates which better describe the true values of DNA methylation level throughout the genome. We propose an experimental scenario for evaluating the true relationship in a high-throughput setting and a model-based analysis to predict the absolute and relative DNA methylation levels. We successfully applied this model to evaluate DNA methylation status of normal human melanocytes compared to a melanoma cell strain. Despite the low resolution typical of methods based on immunoprecipitation, we show that model-derived estimates of DNA methylation provide relatively high correlation with measured absolute and relative levels, as validated by bisulfite genomic DNA sequencing. Importantly, the model-derived DNA methylation estimates simplify the interpretation of the results both at single-loci and at chromosome-wide levels.
The MEDME R library as well as installation instructions and a PDF tutorial are available online at the website below.

overall_design:
MeDIP enrichment for two replicated hybridization of fully methylated genomic DNA are available for the calibration of the model with MEDME. The model can be therefore applied on a real-life dataset. MeDIP enrichment is available for Normal melanocytes (NBMEL) as well as melanoma strain (YUSAC2) (2 replicates each).
For each sample the Absolute and Relative Methylation Scores estimated by MEDME are available.

Expression data for the NBMEL sample is available too, for the evaluation of the effectiveness of the new DNA methylation estimates. Indeed, promoter methylation is known to be associated with transcriptional repression of the down-stream gene.


title: A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies

status: Public on Oct 16 2008

first: Timothy

last: Nelson

department: MCDB

summary:
Whole-genome transcriptional profiles of rice cell types, isolated by laser-capture microdissection. The first comprehensive multi-organ cell type transcriptome atlas from a plant, with 40 distinct cell types from several developmental stages of seeds, shoots and roots, reveals cell-specific promoter motifs, interaction partner candidates, hormone response centers, and other previously unrecognized cellular properties and patterns.

Keywords: Cell type comparison

overall_design:
For GSM329492-GSM329659: Microarray experiments for 40 cell types and two organs were organized using a common reference statistical design, to assure data for all cells could be normalized for cross-comparison. In this design, the hybridization for each cell type sample included a second probe made from an RNA sample that served as a Common Reference, identically aliquoted from a single preparation, for all hybridizations, and including dye swaps for the two probe labels. The Common Reference RNA for all hybridizations was prepared from rice suspension cultures, selected as an undifferentiated cell type that expresses a relatively high percentage of its genome. Although this is an abundant source of RNA, the Common Reference RNA was amplified and labeled in identical fashion to other probes. At least four independent biological replicates for each cell type were collected and profiled; only microarray data sets with r >= 0.9 were retained. A dye swap was included, in which each cell type sample and the Common Reference were labeled with Cy3 and Cy5, respectively, on the two replicates and the reverse for two replicates.

For GSM588319-GSM588370: We analyzed 4 replicates for each cell type and compared with cultured cells. Each replicate contains 2 slides.

first: Yuling

last: Jiao

first: Lori

last: Tausta

first: Neeru

last: Gandotra

first: Ning

last: Sun

first: Nicole

middle: K

last: Clay

first: Tie

last: Liu

first: Teresa

last: Ceserani

first: Meiqin

last: Chen

first: Ligeng

last: Ma

first: Matthew

last: Holford

first: Hongyu

last: Zhao

first: Xing-Wang

last: Deng


title: Comprehensive assessment of the regulons controlled by the FixLJ-FixK2-FixK1 cascade in Bradyrhizobium japonicum

status: Public on Aug 21 2008

city: Zurich

first: Stefan

last: Zoller

address: Universitatstrasse 16

department: Genetic Diversity Centre

institute: ETH Zurich

summary:
Genome-wide transcription profiling of B. japonicum fixJ, fixK2, and fixK1 mutant strains which were grown in free-living microoxic condition, and in the case of the fixK1 mutant also in anoxic condition. Transcriptomes of nodules from soybean plants infected with the fixJ or fixK2 mutant strains were investigated, too. This study includes also the expression data of a nifA mutant grown in free-living anoxic condition. Samples from GSM210242 to GSM210286 in GEO record number GSE8478 need to be considered for the analyses.

Keywords: gene regulation, genetic modification, growth conditions, symbiosis

sample_organism:
Bradyrhizobium japonicum
Glycine max

overall_design: Comparison analysis of the B. japonicum fixJ, fixK2 and fixK1 mutants and the wild type grown under different oxygen conditions in culture and during symbiosis with soybean.

first: S

last: Mesa

first: F

last: Hauser

first: M

last: Friberg

last: Malaguti

first: H

last: Fischer

last: Hennecke


title: A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains

city: Canberra

first: Andrew

last: Spriggs

state: ACT

address: GPO Box 1600

department: Bioinformatics Group

country: Australia

institute: CSIRO Plant Industry

summary:
Endogenous small RNAs, including microRNAs (miRNAs) and short-interfering RNAs (siRNAs), function as posttranscriptional or transcriptional regulators in plants. miRNA function is essential for normal development and therefore likely to be important in the growth of the rice grain. To investigate the likely roles of miRNAs in rice grain development we carried out deep sequencing of the small RNA populations of rice grains. A total of 96,091 (including 23,867 reads from vegetative tissues) and 5,379,724 small RNA sequences that are longer than 17nt were generated. Approximately 94% of these small RNAs were 20-24nt in length. The majority of the small RNAs were singletons, indicating that rice genome has a very complex small RNA population, which is harder to be saturated. From these smal RNA sequences we found representatives of all 20 conserved plant miRNA families and evidence for changes in expression of miRNAs during rice grain development. Using an approach based on the presence of the miRNA and miRNA* sequences, we identified 51 novel, non-conserved rice miRNA families expressed in grains with functionally diverse predicted target genes. miRNA-guided cleavage was confirmed for a number of targets genes including ones with roles in sugar signalling and restoration of cytoplasmic male sterility. We identified a likely mirtron, indicating that plants can also use spliced introns as a source of miRNAs. Our sequencing results revealed four TAS3 loci; these all contain dual miR390 sites of which only the 3? site is cleaved. We also found a miRNA-like long hairpin generating phased 21nt small RNAs, strongly expressed in developing grains and show that these small RNAs act in trans to cleave target mRNAs.
Keywords: high throughput pyrosequencing, small RNA, microRNA, grain development, rice

overall_design: Small RNA populations of shoots and roots of 7 days old seedlings, and 1-5 and 6-10 days after fertilization grains were determined using high throughput sequencing technology. The abundance of known miRNAs were compared based on the number of sequence reads. To give a whole picture of the rice small RNA populations and to reflect un-biasly the sequencing results, small RNAs that are longer than 17nt no matter whether or not they matched with the rice genome were included in this submitted dataset. The unmatched sequences may be derived from un-sequenced regions or sequencing errors.

first: Qian-Hao

last: Zhu

first: Chris

last: Helliwell

first: Frank

last: Gubler

first: Longjiang

last: Fan

first: Louisa

last: Matthew

first: Gavin

last: Kennedy


title: Neural fate stabilization (moody-affy-xenop-315103)

status: Public on Apr 12 2008

city: Phoenix

first: Winnie

last: Liang

state: AZ

address: 445 N. Fifth Street

institute: Translational Genomics

summary:
The goal of the current NS23158 funding is to understand how neural fate-stabilizing (NFS) genes function in order to provide fundamental knowledge about their potential roles in neural stem cell development.  Neural fate-stabilization is characterized by the expansion of the neural plate shortly after the presumptive neural ectoderm has been induced by the repression BMP signaling.  During this time period a number of early-expressed NFS genes are expressed, each of which expands the neural plate in gain-of-function assays.  But, we do not know how these genes are related to one another or whether they are arranged in a linear gene pathway or multi-path hierarchy.  A key aim of this grant is to elucidate the relationship of foxD5, an early-expressed NFS gene that we cloned, to other NFS-genes. We propose to greatly enhance this analysis by utilizing DNA microarray analyses to identify unsuspected and novel target genes.
What is the function of foxD5 in neural fate-stabilization, and how does it specifically relate to other genes in the early-expressed group?  In the parent grant we proposed to study the relationship of early-expressed NFS genes to one that we cloned, FoxD5 (Sullivan et al 2001).  FoxD5 expands the neural plate and holds it in an immature state.  In the original grant, we proposed to study whether foxD5 activates or suppresses the expression of 6 known early-expressed NFS genes, using PCR and in situ hybridization of animal caps and whole embryos.  Herein we propose to use DNA microarray technology to reveal a much broader spectrum of potential target genes.
FoxD5, a fork-head transcription factor which is expressed in the early neuroectoderm, is a key regulator of neural plate fate during expansion of the neural plate.  Being a newly cloned gene, we hypothesize that we will identify numerous downstream targets of FoxD5, by the proposed microarray analyses.  This information will allow us to study their function by gain- and loss-of function studies in the whole embryo.
Animal cap (AC) explants are a naïve embryonic ectoderm that is removed from embryonic signaling centers.  They allow one to perform gene induction assays in the absence of confounding growth factors, and thus are ideal for identifying downstream targets of transcription factors.  In this experiment we will take a gain-of-function approach to identify which genes are induced/repressed by foxD5. FoxD5 mRNA (200pg) will be injected at the 2-cell stage, and embryos cultured until stage 8, at which time the zygotic genome begins transcription.  AC explants will be cut from the embryos and cultured in Normal Amphibians Medium.  When ACs reach stage 10.5, an early step in neural ectoderm specification, they will be snap frozen in liquid nitrogen and RNA purified according to the Qiagen Rneasy Protect kit protocols.  Our preliminary experiments indicate that 8-10ug of total RNA can be recovered with high purity from ~150 ACs with this method.  As controls, RNA will be purified from uninjected animal caps derived from sibling embryos.  If foxD5 represses NFS gene expression, it will not be detected in the above experiment because NFS genes are not expressed in AC explants in the absence of neural induction.  Therefore, we will repeat the experiment but additionally treat the ACs (FoxD5-injected and sibling uninjected) at stage 8 with Noggin protein (R&D Systems) to induce neural ectoderm.  For all four sample sets, RNA will be reverse transcribed, amplified and biotinylated using the NuGEN Ovation kit.
Keywords: neural fate, FoxD5, Noggin

first: Sally

last: Moody


title: Epigenetic events in early breast cancer

status: Public on Jun 05 2008

city: St. Lucia

first: Milena

last: Gongora

state: QLD

address: bld 80, services rd

institute: Institute for Molecular Biosciences

summary:
Subpopulations of primary Human Mammary Epithelial Cells (HMEC) have the unique ability to escape a period of growth arrest and continue to proliferate.  These cells, called post-selection or variant cells (vHMEC), share features with premalignant breast cancer lesions, including p16INK4A promoter hypermethylation.  Epigenetic silencing of tumour suppressor genes through DNA methylation and histone modification is an early event in tumorigenesis. One of the main challenges is to find genes or gene pathways that are commonly silenced to provide early epigenetic diagnostic and therapeutic cancer targets. To identify very early epigenetic events that occur in breast cancer, we used microarrays to screen for gene pathways that were suppressed in post-selection HMECs, but reactivated after treatment with the demethylation agent 5-Aza-2’-deoxycytidine (5-Aza-dC).  We found several members of the Transforming Growth Factor Beta (TGFb) signalling pathway (THBS1, TGFb2, TGFb R1 & TGFb R2) were consistently down-regulated in the post-selection HMEC population. Gene suppression was not associated with DNA methylation but was associated with chromatin remodelling, involving a decrease in histone H3 lysine 27 (H3K27) tri-methylation and an increase in histone H3 lysine 9 (H3K9) di-methylation and H3K9 de-acetylation.  Similar epigenetic repression was also identified MDAMB453 breast cancer cells and in breast tumour samples.  These results demonstrate for the first time that TGFb2, its receptors TGFb R1 & TGFb R2 and activator THBS1 are concordantly suppressed early in breast carcinogenesis by repressive histone modifications and indicate that the TGFb signalling pathway is a novel target for gene activation by epigenetic therapy.
Keywords: cell differentiation, breast cancer, gene silencing, epigenetics

overall_design: Two-colour reference design was used.  Two biological replicates were included in the study - Bre60 and Bre-80.  Each cell line was sampled at pre-selection stage, post-selection stage and post AZA treatment.  The post-selection stage was chosen as the reference sample, and a control post-post hybridisation included to assess variability across the assay.

first: Rebecca

last: Hinshelwood

first: Lily

last: Huschtscha

first: John

last: Melki

first: Clare

last: Stirzaker

first: Timothy

last: Ravasi

first: Christine

last: Wells

first: David

last: Hume

first: Roger

last: Reddel

first: Susan

middle: J

last: Clark


title: Expression data from barley maturing and germinating grains

status: Public on Dec 20 2007

city: Potsdam

first: Björn

last: Usadel

address: Am Mühlenberg 1

country: Germany

institute: Max Planck Institute of Molecular Plant Physiology

summary:
Plant seeds prepare for germination already during seed maturation. We performed a detailed transcriptome analysis of barley grain maturation, desiccation and germination in two tissue fractions (endosperm/aleurone = e/a and embryo = em) using the Affymetrix barley1 chip.
Keywords: time course

overall_design: Barley developing and germinating seeds were harvested at different time points after flowering (developing) and imbibition (germinating). To further disseect the influence of different tissues, seeds were dissecte and tissues were analyzed individually.

first: Nese

last: Sreenivasulu

first: Bjoern

first: Andreas

last: Winter

first: Volodymyr

last: Radchuk

first: Uwe

last: Scholz

first: Niels

last: Stein

first: Winfriede

last: Weschke

first: Marc

last: Strickert

last: Close

first: Mark

last: Stitt

last: Graner

first: Ulrich

last: Wobus


title: Global consequences of phosphatidylcholine reduction in Bradyrhizobium japonicum

status: Public on Apr 17 2008

first: Stefan

last: Zoller

summary:
Analysis of a Bradyrhizobium japonicum pmtA mutant. PmtA catalyzes the first of three consecutive methylation reactions leading to phosphatidylcholine (PC) formation in B. japonicum. Disruption of the pmtA gene results in a significantly reduced PC content causing a defect in symbiosis with the soybean host. This study provides the first insight into global transcriptomic changes of a bacterial phosphatidylcholine biosynthesis mutant. Cells of the pmtA mutant and the wild type were grown to mid-exponential phase in full medium (PSY) under aerobic culture conditions.
Keywords: genetic modification

overall_design: Comparative analyis of the B. japonicum pmtA mutant and the wild type grown under aerobic culture conditions.

first: S

last: Hacker

first: A

last: Lindemann

last: Mesa

first: G

last: Pessi

last: Fischer

first: H

last: Hennecke

first: F

last: Narberhaus


title: Neural fate stabilization (moody-affy-xenop-317980)

first: Winnie

last: Liang

summary:
The goal of the current NS23158 funding is to understand how neural fate-stabilizing (NFS) genes function in order to provide fundamental knowledge about their potential roles in neural stem cell development. Neural fate-stabilization is characterized by the expansion of the neural ectoderm shortly after it has been induced by the repression BMP signaling. During this time period a number of early-expressed NFS genes are expressed, each of which expands the neural plate in gain-of-function assays. But, we do not know how these genes are related to one another or whether they are arranged in a linear gene pathway or multi-path hierarchy. One aim of this grant is to elucidate the relationship of Six1, an early-expressed NFS gene that we cloned, to other NFS-genes. We propose to greatly enhance this analysis by utilizing DNA microarray analyses to identify unsuspected and novel target genes.
What is the function of Six1 in establishing the lateral neural ectoderm, that gives rise to the placodes and how does it specifically relate to other NFS-genes? In the parent grant we proposed to study the relationship of NFS genes to one that we cloned, Six1 (Brugmann et al., 2004). Six1 expands the placodal ectoderm and holds it in an immature state. In the original grant, we proposed to study whether Six1 activates or suppresses the expression of 6 known lateral NFS genes, using PCR and in situ hybridization of animal caps and whole embryos. Herein we propose to use DNA microarray technology to reveal a much broader spectrum of potential target genes.
Six1, a sine oculis transcription factor which is expressed in the early placodal ectoderm that gives rise to the cranial PNS, is a key regulator of neural placodal fate (Brugmann et al., 2004, Development). Being a newly cloned gene, we hypothesize that we will identify numerous downstream targets of Six1 by the proposed microarray analyses. This information will allow us to study their function by gain- and loss-of function studies in the whole embryo.
Animal cap (AC) explants are a naïve embryonic ectoderm that is removed from embryonic signaling centers. They allow one to perform gene induction assays in the absence of confounding growth factors, and thus are ideal for identifying downstream targets of transcription factors. In this experiment we will take a gain-of-function approach to identify which genes are induced/repressed by Six1. Six1 mRNA (400pg) will be injected at the 2-cell stage, and embryos cultured until stage 8, at which time the zygotic genome begins transcription. AC explants will be cut from the embryos and cultured in Normal Amphibians Medium. When ACs reach stage 10.5, an early step in neural ectoderm specification, they will be cultured in 200ng/nl Noggin protein (R&D Systems) to induce the expression of neural plate genes.  When ACs reach stage 16, at which time the placodes have formed, they will be snap frozen in liquid nitrogen and RNA purified according to the Qiagen Rneasy Protect kit protocols. Our preliminary experiments indicate that 8-10ug of total RNA can be recovered with high purity from ~150 ACs with this method. As controls, RNA will be purified from uninjected animal caps, also treated with Noggin, derived from sibling embryos.  We will repeat the experiment 5 times (total of 10 samples).  Extracted RNA will be evaluated for quality, reverse transcribed into cDNA, amplified, fragmented and then biotinylated using the NuGEN Ovation Biotin System kit.
Keywords: neural fate, noggin, Six-1

first: Sally

last: Moody


title: Human embryonic stem cells: media/growth factor effects and differentiation timecourse

status: Public on Mar 12 2008

city: Münster

first: Boris

last: Greber

address: Röntgenstraße 20

department: Cell and Developmental Biology

institute: Max Planck Institute for Molecular Biomedicine

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

overall_design: Refer to individual Series


title: Gene regulation in brain, eye and heart of sighted C3H mice

status: Public on Dec 01 2007

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum München GmbH

summary: It is unclear what role vision plays in guiding mouse behaviour, since the mouse eye is of comparably low optical quality, and mice are considered to rely primarily on other senses. All C3H substrains are homozygous for the Pde6b(rd1) mutation and get blind by weaning age. To study the impact of the Pde6b(rd1) mutation on mouse behaviour and physiology, sighted C3H (C3H.Pde6b+) and normal C3H/HeH mice were phenotyped for different aspects. We confirmed retinal degeneration 1 in C3H/HeH mice, and the presence of a morphologically normal retina as well as visual ability in C3H.Pde6b+ mice. However, C3H.Pde6b+ mice showed an abnormal retinal function in the electroretinogram response, indicating that their vision was not normal as expected. C3H.Pde6b+ mice showed reduced latencies for several behaviours without any further alterations in these behaviours in comparison to C3H/HeH mice, suggesting that visual ability, although impaired, enables earlier usage of the behavioural repertoire in a novel environment, but does not lead to increased activity levels. These results emphasize the importance of comprehensive behavioural and physiological phenotyping.

overall_design: RNA expression profiling of eye, brain and heart of six male animals of the C3H seh mutant mouse line. In total 32 chip hybridizations including biological and experimental replicates (colour flip) were performed. The data analysis and various statistical methods detected several genes differentially regulated between mutant and reference tissues in all analyzed organ types.

first: Johannes

last: Beckers

first: Marion

last: Horsch

first: Jochen

last: Graw


title: Whole genome DNAse hypersensitivity in human CD4+ T-cells

status: Public on Nov 19 2007

first: Sean

last: Davis

address: 37 Convent Drive, Room 6138

institute: National Cancer Institute

summary:
Mapping DNaseI hypersensitive (HS) sites is an accurate method of identifying the location of genetic regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions.  We employed whole genome tiled array strategies to identify DNaseI HS sites within human primary CD4+ T cells.
Keywords: whole genome tiling array

overall_design: DNAse hypersensitivity from two biologic replicates of CD4+ T-cells were hybridized to a whole-genome tiling array set (38 arrays each) and compared to the input DNA from the same samples.

first: Alan

last: Boyle

first: Hennady

last: Shulha

first: Paul

last: Meltzer

first: Zhiping

last: Weng

first: Terrence

last: Furey

first: Gregory

middle: E

last: Crawford


title: Comparison of B. japonicum mutant strain 613 induced versus wild type uninduced

status: Public on Oct 15 2007

city: Dresden

first: Michael

last: Göttfert

address: Helmholtzstrasse 10

department: Biology

institute: Dresden University of Technology

summary:
An initializing step in the rhizobia-legume symbiosis is the secretion of flavonoids by plants that leads to the expression of nodulation genes in rhizobia. Here we report the genome-wide transcriptional response of Bradyrhizobium japonicum to genistein, an isoflavone secreted by soybean. About 100 genes were induced in the wild type. This included all nod box-associated genes, the flagellar cluster and several genes that are likely to be involved in transport processes. To elucidate the role of known regulators, we analysed mutant strains. This revealed that the two-component response regulator NodW is essential for induction of almost all genistein-inducible genes, with the exception of 8 genes. The phenotype of the nodW mutant could be partially suppressed by overexpression of NwsB, which is also a two-component response regulator. These data indicate that genistein has a much broader function than mere induction of nod genes.
Keywords: transcriptional analysis, genistein induction

overall_design: 3 biological replicates of wild type (uninduced) and mutant strain 613 (nodW mutant; induced with genistein) were compared.

first: Kathrin

last: Lang

first: Andrea

last: Lindemann

first: Felix

last: Hauser


title: Response of Bradyrhizobium japonicum wild type and mutant strains to genistein


title: Comparison of B. japonicum 110spc4 (wild type) induced versus uninduced

overall_design: 3 biological replicates of wild type uninduced and wild type induced with genistein were compared.


title: LmrR Lactococcus lactis MG1363

status: Public on Oct 04 2007

first: Anne

middle: de

last: Jong

address: Nijenborgh 7

department: Molecular Genetics

institute: university of Groningen

summary:
Comparison of L. lactis NZ9000 ΔlmrR versus L. lactis NZ9000 wild type
Keywords: Transcription profiling

sample_organism: Lactococcus cremoris subsp. cremoris MG1363

overall_design: Comparison between strain lacking transcriptional regulator LmrR of major Lactococcal mdr transporter LmrCD, and wild type parental strain.

first: Herfita

last: Agustiandari

first: Jacek

last: Lubelski

first: Bart

last: van den Berg van Saparoea

first: Oscar

last: Kuipers

first: Arnold

last: Driessen


title: Comparison of B. japonicum mutant strain Δ901 induced versus wild type uninduced

overall_design: 3 biological replicates of wild type (uninduced) and mutant strain Δ901 (overexpressed nwsB; induced with genistein) were compared.


title: Global gene expression analysis of the phytopathogen Xylella fastidiosa at low iron availability

status: Public on Sep 01 2007

city: Ribeirao Preto

first: Ricardo

last: Vêncio

state: SP

address: Av. Bandeirantes, 3900

department: Computing and Mathematics Department

country: Brazil

institute: Universidade de São Paulo

summary:
Xylella fastidiosa is the etiologic agent of a wide range of plant diseases including citrus variegated chlorosis (CVC), a major threat to the Brazilian citrus industry. Genome sequences of several strains of this phytopathogen are accessible, enabling large-scale functional studies. Transcript levels in different iron availabilities were assessed with DNA microarrays representing 2608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c. When treated with the iron chelator 2,2-dipyridyl, 193 CDS were considered as up-regulated and 216 as down-regulated. In the presence of 100uM of ferric pyrophosphate, 218 and 256 CDS were considered as up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription - quantitative PCR that showed a Pearson correlation of 0.77 with array results. The CDS differentially expressed upon the iron concentration shift participate in diverse cellular functions. Many CDS involved with regulatory functions, pathogenicity and cell structure, were modulated in both conditions tested suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to pili/fimbriae functions. We also investigated the contribution of the ferric uptake regulator Fur to the iron regulon of X. fastidiosa. The promoter regions of strain 9a5c genome were screened for putative Fur boxes and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron regulon of X. fastidiosa and present novel evidence for iron regulation of pathogenicity determinants.
Keywords: stress response; response to iron-depleted condition

overall_design: Direct comparison between low iron content (200uM 2,2-dipyridyl) and control condition. Hybridizations are dye-swaped. There are 2 biological replicates (independent harvest) and 2 technical replicates of each array (L - left and R - right).


title: New target genes controlled by the Bradyrhizobium japonicum two-component regulatory system RegSR

status: Public on Nov 15 2007

first: Stefan

last: Zoller

summary:
Analysis of a mutant in the Bradyrhizobium japonicum response regulator RegR. RegR is known to control expression of the gene encoding the key regulator of nitrogen fixation NifA. This study provides insights into the RegR regulon under free-living conditions and during symbiosis. Cells of the regR mutant and the wild type were grown to mid-exponential phase in full medium (PSY) under different oxygen conditions (aerobic and microaerobic) in culture. Nodules from soybean plant infected with the regR mutant or the wild type were collected 13 and 21 days post inoculation To determine genes under control of RegR during symbiosis. This study includes also the samples GSM210238 to GSM210286.
Keywords: genetic modification, time course, growth conditions

overall_design: Comparison analyis of the B. japonicum regR mutant and the wild type grown under different oxygen conditions in culture and during symbiosis with soybean.

first: A

last: Moser

first: G

last: Pessi

first: F

first: H

last: Hennecke

middle: M

last: Fischer


title: Mapping the C. elegans non-coding transcriptome with a whole genome tiling microarray

status: Public on Sep 07 2007

city: Buffalo

first: Tao

last: Liu

state: NY

address: Elm and Carlton St

department: Biostatistics and Bioinformatics

institute: Roswell Park Comprehensive Cancer Institute

summary:
The number of annotated protein coding genes in the genome of Caenorhabditis elegans is similar to that of other animals, but the extent of its non-protein coding transcriptome remains unknown. Expression profiling on whole genome tiling microarrays applied to a mixed stage C. elegans population verified the expression of 71% of all annotated exons. Only a small fraction (11 %) of the polyadenylated transcription is non-annotated, and appears to consist of approximately 3200 missed or alternative exons and 7800 small transcripts of unknown function (TUFs). Almost half (44%) of the detected transcriptional output is non-polyadenylated and probably not protein coding, and of this 70% overlap the boundaries of protein coding genes in a complex manner. Specific analysis of small non-polyadenylated transcripts verified 98% of all annotated small ncRNAs, and suggested that the transcriptome contains about 1,200 small (<500 nt) unannoted non-coding loci. After combining overlapping transcripts, we estimate that at least 70% of the total C. elegans genome is transcribed.
Keywords: RNA fractions

overall_design: RNA was extracted from mixed stage wild-type N2 strain worms cultivated at 20oC according to the Trizol (Invitrogen) protocol. Small RNAs (< 500nt, SNPA sample) were isolated using a Qiagen tip (Qiagen), and the Poly(A) Purist MAG (Ambion) and MicrobExpress kits (Ambion) were adapted to remove remaining mRNAs and rRNAs (Deng et al. 2006). The enriched ncRNA pool was cloned using an adaptor-mediated library construction protocol. RNAs were dephosphorylated with calf intestine alkaline phosphatase (Fermentas), then ligated to the 3’-adaptor (3AD) oligonucleotide by T4 RNA ligase (Fermentas) (He et al. 2006). Polyadenylated RNA (PA sample) was isolated from total RNA using the Poly(A) Purist MAG kit (above). Non-polyadenylated RNA (NPA sample) was prepared by removing polyadenylated RNA using the Poly(A) Purist MAG kit and rRNA using the MicrobExpress kit.

first: Housheng

last: He

first: Jie

last: Wang

first: Geir

last: Skogerbø

first: Runsheng

last: Chen


title: WT (Fis+) strain

status: Public on Aug 15 2007

city: Albany

first: Robert

last: Osuna

address: 1400 Washington Avenue

institute: University at Albany

summary:
Fis is a nucleoid-associated protein in E. coli that is abundant during early logarithmic growth in rich medium but is in short supply during stationary phase.  Its role as a transcriptional regulator has been demonstrated for an increasing number of genes.  In order to gain insight into the global effects of Fis on E. coli gene expression during different stages of growth in rich medium, DNA microarray analyses were conducted in fis and wild type strains during early log, mid log, late log, and stationary growth phases.
We used microarrays to detail the global impact of Fis on gene expression in Escherichia coli
Keywords: time course

overall_design: E.coli cells were were grown and samples were  taken at different times during growth: 90 min (early logarithmic phase), 150 min (mid-logarithmic phase), 240 min (late logarithmic phase), and 360 min (early stationary phase) for RNA extraction and hybridization on Affymetrix microarrays.  This was done in triplicate and the raw data was analyzed using Microarray Analysis Suite version 5.0 (Affymetrix).

first: Meranda

middle: D

last: Bradley

first: Michael

last: Beach

first: Timothy

last: Pratt


title: Global gene expression analysis of the phytopathogen Xylella fastidiosa at high iron availability

summary:
Xylella fastidiosa is the etiologic agent of a wide range of plant diseases including citrus variegated chlorosis (CVC), a major threat to the Brazilian citrus industry. Genome sequences of several strains of this phytopathogen are accessible, enabling large-scale functional studies. Transcript levels in different iron availabilities were assessed with DNA microarrays representing 2608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c. When treated with the iron chelator 2,2-dipyridyl, 193 CDS were considered as up-regulated and 216 as down-regulated. In the presence of 100uM of ferric pyrophosphate, 218 and 256 CDS were considered as up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription - quantitative PCR that showed a Pearson correlation of 0.77 with array results. The CDS differentially expressed upon the iron concentration shift participate in diverse cellular functions. Many CDS involved with regulatory functions, pathogenicity and cell structure, were modulated in both conditions tested suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to pili/fimbriae functions. We also investigated the contribution of the ferric uptake regulator Fur to the iron regulon of X. fastidiosa. The promoter regions of strain 9a5c genome were screened for putative Fur boxes and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron regulon of X. fastidiosa and present novel evidence for iron regulation of pathogenicity determinants.
Keywords: stress response; response to iron-replete condition

overall_design: Direct comparison between high iron content (100uM ferric pyrophosphate) and control condition. Hybridizations are dye-swaped. There are 2 biological replicates (independent harvest) and 2 technical replicates of each array (L - left and R - right).

first: Paulo

last: Zaini


title: Drosophila MSL complex selectively identifies active genes on the male X chromosome

status: Public on Aug 01 2007

city: Boston

first: Peter

last: Park

address: 10 Shattuck St

department: Center for Biomedical Informatics

institute: Harvard Medical School

summary:
X-chromosome dosage compensation in Drosophila requires the male-specific lethal (MSL) complex, which up-regulates gene expression from the single male X chromosome. Here, we define X-chromosome-specific MSL binding at high resolution in two male cell lines and in late-stage embryos. We find that the MSL complex is highly enriched over most expressed genes, with binding biased toward the 3' end of transcription units. The binding patterns are largely similar in the distinct cell types, with ~600 genes clearly bound in all three cases. Genes identified as clearly bound in one cell type and not in another indicate that attraction of MSL complex correlates with expression state. Thus, sequence alone is not sufficient to explain MSL targeting. We propose that the MSL complex recognizes most X-linked genes, but only in the context of chromatin factors or modifications indicative of active transcription. Distinguishing expressed genes from the bulk of the genome is likely to be an important function common to many chromatin organizing and modifying activities.
Keywords: ChIP-chip

overall_design:
Comparison of MSL binding in two male cell lines (SL2 and Clone8) and in embryos:
Tiling arrays containing 388,000 probes were designed based on FlyBase 3.2. Chromosome X and 19.6 Mb of chromosome 2L were tiled; chromosome 2L was useful as a control to verify that the features identified on the X are specific to it. For SL2 cells, two independent experiments were performed. Regions identified as bound in initial dye-swap replicates for the two SL2 experiments were very similar, and no further dye-swaps were performed. Two experiments and a dye-swap for one of them were used in the SL2 analysis. For Clone 8 cells and embryos, two experiments were performed for each cell type. Each experiment involved two arrays, one serving as a mock control in which the same protocol was followed in the absence of the TAP-tagged MSL complex. In total, seven log-ratio signals derived from 14 arrays were used: three for SL2 cells, two for Clone 8 cells, and two for embryos. The reproducibility between independent experiments within a cell type was excellent and care was taken to insure that the analysis is not biased by the uneven number of samples. In the embryo data, for instance, 776 and 766 clusters were identified in the two independent experiments and 757 were shared between the two.

first: Mitzi

middle: I

last: Kuroda


title: MSL complex is attracted to genes marked by H3K36 trimethylation using a sequence-independent mechanism

status: Public on Sep 20 2007

summary:
In Drosophila, X chromosome dosage compensation requires the male-specific lethal (MSL) complex, which associates with actively transcribed genes on the single male X chromosome to upregulate transcription approximately 2-fold. We found that on the male X chromosome, or when MSL complex is ectopically localized to an autosome, histone H3K36 trimethylation (H3K36me3) is a strong predictor of MSL binding. We isolated mutants lacking Set2, the H3K36me3 methyltransferase, and found that Set2 is an essential gene in both sexes of Drosophila. In set2 mutant males, MSL complex maintains X specificity but exhibits reduced binding to target genes. Furthermore, recombinant MSL3 protein preferentially binds nucleosomes marked by H3K36me3 in vitro. Our results support a model in which MSL complex uses high-affinity sites to initially recognize the X chromosome and then associates with many of its targets through sequence-independent features of transcribed genes.
Keywords: ChIP-chip

overall_design: ChIP-chip experiments were performed on custom Nimblegen arrays (GPL5636).  Each array contained 388,000 oligonucleotide probes covering all of the X and the 2L chromosomes, with a 100 bp resolution (50mer probes with 50 bp gaps).  The design was based on FlyBase 3.2.  For the superspreading experiments, an additional array was used that contains the entire X chromosome and 3R (GPL5660).  MSL complex binding sites on both arrays are the same and signal on the 3R chromosome was at background level.


title: Fis KO strain


title: Whole genome tiling path array CGH analysis of segmental copy number alterations in cervical cancer cells

status: Public on Jun 12 2007

city: Bethesda

first: William

middle: Wallace

last: Lockwood

address: 50 South Drive

institute: National Human Genome Research Institute

type: Genome variation profiling by genome tiling array

summary:
Cervical cancer is the second most common malignancy in women worldwide, with high risk subtypes of human papillomavirus (HPV) constituting the major etiological agent.  However, only a small percentage of women infected by the virus develop disease suggesting that additional host genetic alterations are necessary for disease progression.  In this study we examined the genomes of a panel of commonly used model cervical cancer cell lines using a recently developed whole genome tiling path array for CGH analysis.  Detailed analysis of genomic profiles enabled the detection of many novel aberrations which may have been missed by conventional cytogenetic methods.  In total, 27 minimal regions of recurrent copy number alteration were identified that are potentially involved in tumorigenesis. Interestingly, fine mapping of the 3q gain, which is associated with the progression of precursor lesions to invasive cervical cancer, identified a minimal region of alteration harboring genes distinct from previous candidates.  Novel regions of gene amplification, including the co-amplification of both the Birc and MMP gene clusters on 11q22, were also evident.  Lastly, characterization of genomic structure at sites of HPV integration identified the copy number gain of host cellular sequences between the viral-host genomic boundaries in both SiHa and SW756, suggesting a direct role for HPV integration in the development of genetic abnormalities that initiate cervical cancer.  This work represents the highest resolution look at a cervical cancer genome to date and offers definitive characterization of the alteration status of these cancer genomes.
Keywords: array CGH, copy number, cervical cancer, genetic alterations, HPV integration, 3q

overall_design: Whole genome tiling path array CGH profiles of 8 Cervical Cancer Cell Lines

middle: W

first: Bradley

middle: P

last: Coe

first: Ariane

middle: C

last: Williams

first: Calum

last: MacAulay

first: Wan

last: Lam


title: E15.5 renal macrophage vs E15.5 total kidney

status: Public on Jul 04 2007

city: St Lucia

first: Darrin

middle: F

last: Taylor

state: Qld

address: Research Rd

department: Genomics and Bioinformatics

institute: Institute for Molecular Bioscience, Uni of Qld

summary:
Direct comparison of FACS sorted EGFP positive renal macrophages vs pooled Total Kidney extract
Keywords: Direct pairwise comparison

overall_design: Pairwise comparison containing a dye swap to eliminate dye bias.

first: Kyra

last: Woods

first: Fiona

last: Rae

first: Tedjo

last: Sasmono

first: Naomi

last: Campanale

first: Dmitry

last: Ovchinnikov

first: Sean

last: Grimmond

first: David

last: Hume

first: Sharon

last: Ricardo

first: Melissa

middle: H

last: Little


title: Effect of surface waters from Eglin Air Force Base on gene expression in fathead minnow (Pimephales promelas)

status: Public on Aug 11 2010

city: Providence

first: Daniel

middle: James

last: Spade

state: RI

address: Box G-E5

department: Pathology and Laboratory Medicine

institute: Brown University

summary: The federally endangered Okaloosa darter (Etheostoma okaloosae) is found almost exclusively on the Eglin Air Force Base in the Choctawhatchee Bay watershed of Florida. Portions of this limited habitat are threatened with erosion of soils, altered hydrology, and impaired water quality. One stream reach in particular, East Turkey Creek, has demonstrated potential water quality problems including poor invertebrate bioassessment scores (IBI), uncharacteristically high conductivity values, and low numbers of Okaloosa darters. General water quality (dissolved oxygen, specific conductance, pH, temperature, and relative turbidity and primary productivity) was characterized in both the potentially impacted East Turkey Creek and a reference stream (Long Creek). Water quality was assessed during a 30 day exposure using passive samplers for both non-polar and polar effluent parameters. Metal loading in the system was assessed via fish tissue burdens in resident Pteronotropis hypseleotris. Additionally, microarray analysis was performed on gonad and liver tissue from fathead minnows, Pimephales promelas, after 48-h exposures to water collected from the two creeks and brought into the laboratory. Gene expression changes were evident at the site below the influence of a wastewater spray field sited along East Turkey Creek, suggesting that anthropogenic compounds in the effluent waters may have affected both liver and testis function and could be related to account the general decrease in populations of the Okaloosa darter.

overall_design: Array hybridizations were performed using a single color design. Four biological replicates consisting of four different individuals were analyzed for each of the treatments (University of Florida control, and each of five sites). Site C was left out of subsequent analysis for publication, due to poor characterization of the site.  Two Samples were left out of the normalization/analysis due to QC failure.  Therefore, there are only 3 replicates of the "testis, site A water, exposed 48 h." and "liver, site E water, exposed 48 h."  groups.

first: Roxana

last: Weil

first: Iris

last: Knoebl

first: John

last: Hemming

first: M

last: Tongue

first: Nancy

last: Szabo

first: Kevin

last: Kroll

first: W

middle: B

last: Tate

last: Denslow


title: Genetic reconstruction of a functional transcriptional regulatory network

status: Public on Apr 01 2007

city: Austin

first: Vishy

last: Iyer

state: TX

address: 2500 Speedway Dr. MBB 3.212

department: Molecular Biosciences

institute: University of Texas at Austin

summary:
Although global analyses of transcription factor binding provide
one view of potential transcriptional regulatory networks,
regulation also occurs at levels distinct from transcription
factor binding. Here, we use a genetic approach to identify
targets of transcription factors in yeast and reconstruct a
functional regulatory network. First, we profiled transcriptional
responses in S. cerevisiae strains with individual deletions of
263 transcription factors. Then we used directed-weighted
graph modeling and regulatory epistasis analysis to identify
indirect regulatory relationships between these transcription
factors, and from this we reconstructed a functional
transcriptional regulatory network. The enrichment of
promoter motifs and Gene Ontology annotations provide
insight into the biological functions of the transcription factors.

Data values are X scores and corresponding p-values derived using an error model.
Keywords: X scores and corresponding p-values derived using an error model

overall_design: We profiled transcriptional responses upon the individual deletion of more than 260 TFs. We used a directed-weighted graph modelling approach and regulatory epistasis analysis to identify indirect regulatory relationships, and thus constructed a refined, functional transcriptional regulatory network.

first: Zhanzhi

last: Hu

first: Patrick

last: Killion

first: Vishwanath

middle: R


title: Chromatin immunoprecipitation (ChIP) assay of CWO protein with Drosophila genome tiling array

status: Public on Apr 24 2007

city: Kobe

first: Rikuhiro

middle: G

last: Yamada

state: Hyogo

address: Minatojima-Minamimachi 2-2-3

department: Center for Developmental Biology

country: Japan

institute: RIKEN

summary:
CWO binding sites were genome-widely searched with Drosophila genome tiling array.

Abstract:
The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNAi system in vivo.  Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix-ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tilling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo.  Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.
Keywords: ChIP-chip

overall_design:
4 Drosophila genome tiling arrays were used as follows:
Array1	Immunoprecipetated with anti-Flag antibody (Biological replicate no.1) for CWO binding signal
Array2	Immunoprecipetated with anti-Flag antibody (Biological replicate no.2) for CWO binding signal
Array3	Immunoprecipetated with anti-V5 antiboy (Biological replicate no.1) for background signal
Array4	Immunoprecipetated with anti-V5 antiboy (Biological replicate no.2) for background signal

first: Akira

last: Matsumoto

first: Maki

middle: U

last: Tadenuma

first: Jerry

last: Houl

first: Kenichiro

last: Uno

first: Takeya

last: Kasukawa

first: Brigitte

last: Dauwalder

first: Taichi

middle: Q

last: Itoh

first: Kuniaki

last: Takahashi

first: Ryu

last: Ueda

first: Paul

last: Hardin

first: Teiichi

last: Tanimura

first: Hiroki


title: Male and female embryonic chicken brains (arnol-affy-chick-345142)

status: Public on Mar 20 2007

first: Winnie

last: Liang

address: 445 N. Fifth Street

institute: Translational Genomics

summary:
The study is relevant to an understanding of the forces that lead to sex differences in the brain.  Many neural and psychiatric diseases affect men and women differently, so the understanding of sex differences in brain function impacts on our understanding of why the male and female brain differ in their susceptibility to disease.
Using Affymetrix chicken arrays, we will measure the gene expression in male and female embryonic chicken brain.
Gene expression differs in the male and female embryonic chicken brain.
20 male and 20 female chicken embryos will serve as source of brain tissue.  In late stage embryos, we will remove the brain tissue and extract total RNA.  Four birds will comprise each individual sample.  Thus, we will have 5 biologically independent male samples, and an equal number of female samples.
Keywords: dose response

first: Arthur

last: Arnold


title: The S. cerevisiae histone demethylase Jhd1 fine-tunes the distribution of H3K36me2

status: Public on May 04 2007

city: Chapel Hill

first: Jason

last: Lieb

address: 203 Fordham Hall

department: Department of Biology

institute: University of North Carolina at Chapel Hill

summary:
Histone methylation plays important roles in the regulation of chromatin dynamics and transcription. Steady state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in S. cerevisiae, is an H3K36-specific demethylase. Here we report further characterization of Jhd1. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and alpha-ketoglutarate as cofactors. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Finally, chromatin immunoprecipitation coupled microarray (ChIP-chip) studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units.
Keywords: ChIP-chip

overall_design: H3K36me2 ChIPs were performed on wild type, jhd1 knockout, and JHD1 overexpression yeast strains.

first: Jia

last: Fang

first: Gregory

middle: J

last: Hogan

first: Gaoyang

first: Yi

last: Zhang


title: Male and female embryonic chicken hearts (arnol-affy-chick-445639)

summary:
The study is relevant to an understanding of the forces that lead to sex differences in the brain and other somatic tissues. Many neural and psychiatric diseases affect men and women differently, so the understanding of sex differences in brain function impacts on our understanding of why the male and female brain differ in their susceptibility to disease.
Using Affymetrix chicken arrays, we will measure the gene expression in male and female embryonic chicken heart, and compare to previous studies of brain and liver.
Gene expression differs in the male and female embryonic chicken heart, liver, and brain.  Higher expression of Z-chromosome genes in males is found in heart, liver and brain.  Comparison of heart and brain will reveal brain-specific patterns of sex differences, and patterns that apply to multiple tissues.
20 male and 20 female chicken embryos will serve as source of heart tissue. In late stage embryos, we will remove the tissue and extract total RNA. Four birds will comprise each individual sample. Thus, we will have 5 biologically independent male samples, and an equal number of female samples.
Keywords: dose response