title: Common and overlapping oncogenic pathways contribute to the evolution of acute myeloid leukemias

status: Public on Jun 27 2011

first: Brian

last: Huntly

address: Wellcome Trust/MRC Building, Hills Road

department: Cambridge Institute for Medical Research

institute: University of Cambridge

summary:
Recently, it has been demonstrated that transcriptionally active leukemia-associated fusion oncogenes alter self-renewal in and generate acute myeloid leukemia (AML) from committed progenitors, linking transformation and self-renewal pathways. AML is a heterogeneous disease, both genetically and biologically, and it is not known whether transformation is mediated by common or overlapping genetic programs downstream of multiple mutations or through the engagement of unique programs downstream of individual mutations. This distinction is important, as the demonstration of common pathways may identify common molecular targets for the treatment of AML.
Here we demonstrate that the ability to alter self-renewal in vitro and in vivo is a more generalized property of leukemia-associated oncogenes. We further demonstrate that disparate leukemia-associated oncogenes initiate early common and overlapping transformation and self-renewal gene expression programs to mediate these effects. Furthermore, elements of these programs can be detected in established leukemia stem cells from an animal model and across a large cohort of patients with differing acute myeloid leukemia (AML) subtypes, where they strongly predict for disease biology. Finally, individual genes from the programs are demonstrated to partially phenocopy the leukemia-associated oncogenes and themselves alter self-renewal in committed murine progenitors and generate AML when expressed in murine bone marrow.

overall_design: The dataset comprises granulocyte monocyte precursors (GMP) transduced with either GFP alone or the fusion oncogenes MOZ-TIF2 or NUP98-HOXA9 or AML1-ETO, as labelled. Early gene expression changes (36 hours after transduction) were analysed between these replicate samples in two separate ways; either in a single two-class comparison of all oncogene transduced GMP with empty-vector transduced GMP or in three separate two-class comparisons comparing each oncogene individually with empty-vector transduced GMP. In the first analysis we identified 1082 genes/1119 probesets whose expression levels significantly differed (FDR level 0.05). There is a loss of self-renewal potential in the transition from the LSK to GMP population and to further enrich for potential self-renewal genes we compared the expression of these genes/probes in sorted normal LSK versus GMP populations. Genes which were not significantly (p<0.05) and similarly differentially expressed between LSK and GMP were excluded, allowing us to prioritize an immediate “leukemia initiation” program of 167 genes (182 probesets). Our second analysis demonstrated differential expression of 5750, 1161, and 5109 genes, when MOZ-TIF2, NUP98-HOXA9 and AML1-ETO transduced GMP were respectively compared with empty-vector transduced GMP (p<0.05). When our “leukemia initiation” signature was compared to the single comparisons, 127/167 (76%) genes overlapped. To assess the evolutionary nature of transcriptional programs critical for leukemia induction and maintenance, the gene expression profiles of replicate functionally validated leukemic stem cells from a MOZ-TIF2 model of leukemia (L-GMP) were then compared to their normal phenotypic counterpart (normal GMP). 2715 genes were differentially expressed between normal and leukemic GMP (p<0.05). Similarities were seen between our immediate pre-leukemic signatures and the gene expression profiles in the overt leukemias. The overlap with the leukemia initiation and leukemia self-renewal signatures was highly significant at 59/167 genes (35%) and 29/91 genes (32%), respectively (both p<0.0001). Gene expression levels were measured using Affymetrix (Santa Clara, CA) GeneChip Mouse Genome 430A 2.0 arrays (22,690 probesets) with hybridisation and washes as per the manufacturers specifications. The starting point for all analyses was the ''.CEL'' files from the MAS5 software. Data was analyzed using the R statistical package bioconductor18. Data quality was assessed using functions in the affy and affyPLM packages and outlier arrays were removed from subsequent analysis. The GCRMA algorithm (ver. 2.4.1) was used to obtain normalized expression estimates. Genes were selected for further analysis on the following basis: genes that had probesets for which the expression value was greater than 60 (which in our study constitute the average background reading of all probesets) and that had a present flag call in at least two of three samples. To detect significant changes in the expression levels, two-sample Welch t-tests (parametric; assuming unequal variances; Benjamini and Hochberg step-up multiple testing correction at a False Discovery Rate <0.05) was applied to the resulting genes.

first: Brynn

middle: T

last: Kvinlaug

middle: J


title: Differential Expression Studies in Murine Splenic Cells During Prion Infection

status: Public on Jul 01 2011

city: Winnipeg

first: Rhiannon

middle: LCH

last: Huzarewich

state: MB

address: 1015 Arlington St.

department: Molecular PathoBiology

country: Canada

institute: Public Health Agency of Canada

summary: DNA microarrays were used to profile gene expression in normal murine splenic cells vs. prion infected splenic cells with the aim of identifying host factors involved in prion propagation and/or useful as biomarkers for early diagnosis.

overall_design:
Two condition experiment, prion infected murine splenic cells vs. normal murine splenic cells: 4 infected biological replicates, 2 control biological replicates, 2 technical replicates (dye-swaps) for each competitive hybridization.

Samples were run on 4 different arrays:
MPB Murine 16K BMAP-MSV printrun 7.1 microarray
MPB Murine 16K BMAP-MSV printrun 9.1 microarray
MPB Murine 32K Oligo printrun 4.1 microarray
Agilent 4x44K Whole Mouse Genome

middle: L

first: Stephanie

middle: A

last: Booth


title: Gene expression profiling in lungs of mice with mesenchyme-specific GR ablation (GRCol1-Cre)

status: Public on Jun 23 2011

city: Houston

first: Scott

middle: Andrew

last: Ochsner

state: TX

address: One Baylor Plaza

department: Molecular and Cellular Biology

country: USA

institute: Baylor College of Medicine

summary: The goal of the experiment was to assay the role of the glucocorticoid receptor (GR) in development of mesenchynmal cells of the lung occuring between the 16 and 18 day of embryonal development.

overall_design: Two time points, E16.5 and E18.5, were analyzed for mutants (GRCol1-Cre) and controls, respectively. For each of the four conditions, five GeneChips were used each representing one animal.

first: Daniel

last: Habermehl

first: Jan

middle: R

last: Parkitna

first: Gunther

last: Schutz


title: Selection of NPC specific Transcription factors

status: Public on Jun 07 2011

city: Tokyo

first: Takafusa

last: Hikichi

address: 1-21-1 Toyama, Shinjuku-ku

country: Japan

institute: National Center for Global Health and Medicine

summary: To construct a candidate pool of NPC specific transcription factor (TF), we selected the genes expressed at relatively high levels in NPCs by comparing them to mixed RNA derived from multiple organs, eliminating housekeeping genes. As result, 160 putative NPC specific TFs were selected.

overall_design: We used three independent NPC lines (NSEB5-2C, NS195R, NS21) established from different ES cell lines (EB5, 195R, 21, respectively). Genes exhibiting relatively higher expression (1.5 fold) in NSEB5-2C and NS195R (or, NSEB5-2C and NS21) were selected by comparing with Mixture (consisting of equal microgram of RNAs from heart, kidney, thymus, spleen, liver, lung, d11 embryo). Gene Ontology (GO) terms ‘DNA binding’ and ‘transcription activity’ were used to select TFs.

first: Shinji

last: Masui


title: Unsupervised hierarchical clustering of iHPCs induced by 9 or 10 TFs

summary: To clarify the gene expression profile of iHep, microarray analysis was performed using iHeps induced by 10 TFs (Foxg1, Lcor, Hnf3b, Hnf4a, Foxo6, Cdx2, Tcf1, Foxa3 ,Tcf2, Onecut1) and 9 TFs (Onecut1 was omitted from 10 TFs). Unsupervised hierarchical clustering indicated that iHep is expressing a global transcriptional profile more similar to that of HPCs rather than that of NPCs, and suggested that TFs present in the pool acted as inducing TFs.

overall_design: HPC (HB1 and HNG2) were established from fetal liver (E13.5) of C57BL6J and STOCK Tg(Nanog-GFP, Puro)1 Yam, respectively. iHeps were induced from NPC (NSBAg2, established from an ES cell line BAg73C2 carrying beta-geo knock-in allele in Afp) using retroviral vectors (pMXs without drug-selection markers) of 9 or 10 transcription factors. Three weeks after the infection, G418 was added and cultured for 1 week before the harvest. NSBAg2 and NSEB5-2C were used for the data of NPC. GSM396240 and GSM336010 were used for the data of ESC.


title: Lgr4/5/6 family members reside in Frizzled-Lrp complexes and mediate signaling by the Wnt agonist Rspondin1

status: Public on Jul 04 2011

city: Dresden

middle: E.

last: Stange

address: Fetscherstr. 74

country: Germany

institute: University Clinic Dresden

summary: The adult stem cell marker Lgr5 and its close relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. Conditional deletion of the two genes in the gut impairs expression of Wnt target genes and results in rapid demise of intestinal crypts, thus phenocopying the effects of Wnt pathway inhibition. By mass-spectrometry, we find that Lgr4 and Lgr5 associate with the endogenous Wnt co-receptors Frizzled-5/7 and Lrp5/6 in HEK293 cells and in colorectal cancer cells. We also find that soluble Rspondin1, a Wnt pathway agonist, specifically binds to endogenous Lgr4 on HEK293 cells. In these cells, soluble Rspondin1 potently enhances canonical Wnt signals initiated by Wnt3A. Removal of Lgr4 does not affect Wnt3A-induced signals, but abrogates the Rspondin1-mediated enhancement of these signals, a phenomenon rescued by re-expression of Lgr4, -5 or -6. Rspondin1 and can be rescued by Wnt pathway activation. Lgr4/5/6 are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble Rspondin proteins.

overall_design: We used two different experimental setups to show that concomitant deletion of Lgr4 and Lgr5 affects the expression of Wnt pathway target genes. First, we determined the downregulated genes after Lgr4/5 deletion in AhCre_Lgr4fl/fl_Lgr5 fl/fl mice on day 1 post-deletion, before any histological changes are apparent in these mice (Lgr4/5 gene signature). Next, we acutely withheld the Wnt agonist Rspondin1 from in vitro crypt organoid cultures and performed differential gene expression before, and 1 day after withdrawal (Rspondin1 withdrawal signature). For better comparison we also deleted the Lgr4 and Lgr5 in vitro in organoids isolated from VillinCre_Lgr4fl/fl_Lgr5 fl/fl mice, and performed microarrays 1 day after deletion in vitro. Furthermore, we used published expression data from Apcfl/fl mice (Apc deletion signature). Deletion of Apc results in the immediate upregulation of Wnt pathway target genes. Comparison of the Lgr4/5 gene signature to both the Rspondin1 withdrawal signature and the Apc deletion signatures using Gene Set Enrichment Analysis (GSEA) showed that the genes deregulated after simultaneous deletion of Lgr4 and -5 are in the majority under the control of the Wnt pathway.

first: Vivian

last: Li

first: Marc

last: van de Wetering

first: Hans

last: Clevers


title: The overexpression of Pax6 affects mesenchymal to epithelial transition (MET) pathway during iPS induction in NPC.

summary: We performed a microarray analysis to compare N31EGFP and N31Pax6 transcriptional profiles 24 h after iPS induction. Among 603 genes whose levels changed greater than two-fold (295 lower, 308 higher), we noticed a 10-fold decrease in the Cdh1 signal in N31Pax6, which was confirmed by reverse transcription-mediated quantitative polymerase chain reaction (RT-QPCR). iPS induction is known to require activation of the mesenchymal to epithelial transition (MET) pathway, which involves Cdh1 upregulation. In fact, Twist1 expression, repression of which is required for MET pathway activation was higher in N31Pax6.

overall_design: Dox-inducible iPS-NPC, N31, was established from the primary iPSC induced from NSEB5-2C by introducing dox-inducible Oct3/4, Sox2, Klf4 and c-Myc. EGFP or Pax6 were overexpressed in N31 by transfecting CAG-EGFP-IP or CAG-Pax6-IP by Tol2 transposon. Gene expression was measured at  24 hours after exposure to doxycycline, when apparent morphological change was observed only in N31EGFP.


title: Systematic identification of transcription factors capable of inducing cell-type-specific transcriptional profiles

status: Public on Jun 10 2011

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

overall_design: Refer to individual Series


title: Unsupervised hierarchical clustering of iNPCs induced by 6 or 5 TFs

summary: To clarify the gene expression profile of iNPC, microarray analysis was performed using iNPCs induced by 6 TFs (Pax6, Hmga2, Etv6, Gatad2b, Nfxl1, and Esx1) and 5 TFs (Esx1 was omitted from 6 TFs). Unsupervised hierarchical clustering indicated that iNPC is expressing a global transcriptional profile more similar to that of NPCs rather than that of MEFs, and suggested that the TFs present in the pool acted as inducing TFs.

overall_design: iNPCs were induced from MEF using 6 or 5 transcription factors. iNPCs were induced from MEF (MEFSH, derived from mice carrying IRES-Hygro in Sox allele) using retroviral vectors (pMXs-IRESNeo) of 6 or 5 transcription factors. Four weeks after the infection, Hygromycin was added and cultured for 1 week before the harvest. NSBAg2 and NSEB5-2C were used for the data of NPC. GSM396240 and GSM336010 were used for the data of ESC.GSM651349 and GSM336011 were used for the data of MEF.


title: Overexpression of each of the 6 interfering TFs and 5 non-interfering TFs

summary: To clarify that the interfering effect in iPS induction was not because of extraordinary gene expression which was caused by overexpression of infected genes, transcriptional profile of the cells infected six genes were analysed using microarray analysis. As result, overexpression of each of the 6 interfering TFs in NSEB5-2C did not compromise transcriptional profile compared with the five non-interfering TFs.

overall_design: The cells were infected by retroviruses (carrying each ORF in pMXs-IRESNeo) at the titre in which around 95% of cells express the transgene. After 24hr of the infection, G418 was added to eliminate non-expressing cells. Each cells were harvested at 72hr after the infection.


title: Effect of FGF15 or FGF19 on mouse liver

city: Dallas

first: Matthew

middle: Joseph

last: Potthoff

address: 6001 Forest Park Rd

department: Pharmacology

institute: University of Texas Southwestern Medical Center

summary: Mouse FGF15 and human FGF19 are orthologous proteins that regulate bile acid metabolism.  However, other hepatic functions of FGF15/19 are not well characterized.

overall_design: We used microarrays to analyze global hepatic gene expression in mice administered FGF15 or FGF19. Total liver RNA was isolated from wild-type mice administered vehicle, purified FGF15 or recombinant FGF19 for 6 hr via a jugular vein injection as described (Inagaki et al., 2005).  Mice did not have access to food throughout the 6 hr treatment, and livers were harvested and immediately frozen in liquid nitrogen.  Microarray analyses were performed using the Mouse Genome 430A 2.0 array.

first: Steven

last: Kliewer

first: David

last: Mangelsdorf


title: Expression data from Pax7+ satellite cell derived tumors in vitro and in vivo

status: Public on Jun 01 2011

city: Durham

first: Jordan

middle: M

last: Blum

state: NC

address: LSRC 450 Research Dr

department: Department of Radiation Oncology

institute: Duke University

summary:
Mouse muscle stem cells, defined as Pax7+ satellite cells, can initiate rhabdomyosarcoma when transformed by oncogenic Kras and concomitant loss of p53.
Mouse Pax7+ satellite cells were transformed in vitro and in vivo utilizing the Cre-ER/loxp system.

overall_design: We wanted to address two major questions: do the in vitro and in vivo tumors cluster together compared to another mouse to another mouse derived soft-tissue sarcoma AND which human soft-tissue sarcoma do the in vivo derived tumors resemble transcriptionally?  Therefore, tumors from cells transformed in vitro and tumors from mice that restrict the oncogenic lesions to Pax7+ satellite cells in vivo were compared to answer these two questions.

first: Zhizhong

last: Kirsch


title: Adipose tissue analysis of wild-type and beta-carotene 15-15'-oxygenase 1 (Bcmo1) ko mice fed under control diet and beta-carotene diet

city: Cleveland

first: Jaume

last: Amengual

state: Ohio

address: 2109 Aderbert Rd

institute: case western reserve university

summary: Cell culture studies indicate that beta-carotene-(BC)-derived apocarotenoid signaling molecules influence the activities of nuclear receptors that regulate many aspects of adipocyte physiology. Here, we analyzed the roles of the two BC metabolizing enzymes, BCMO1 and BCDO2, for this process in mouse models. Wild-type mice converted BC into retinoids and showed reduced body adiposity (by 28%), leptinemia and adipocyte size. Genome wide microarray analysis revealed a generalized decrease of mRNA expression of peroxisome proliferator-activated receptor gamma (PPARgamma) target genes. Consistently, the expression of this key transcription factor for lipogenesis was significantly reduced both on the mRNA and protein levels. This effect of BC was absent in Bcmo1-/- mice that showed increased BCDO2 expression and gamma-10gamma-apocarotenoid production. Our study evidences an important role of BC for the control of body adiposity in mice and identifies Bcmo1 as critical molecular player for the regulation of PPARgamma activity.

overall_design:
The goal of the study was to analyze the putative differential effects of BC accumulation on Bcmo1 ko mice, and BC metabolites in wt mice.
Inguinal adipose tissue gene expression profile comparing adult male and female mice under control diet and beta-carotene (BC) supplemented diet during 14 weeks.
47 mice in total. 23 wild-type (11 male/12 female) and 24 bcmo1 (12 male/12 female) mice, fed on vitamin A diet (1500 IU vit A/kg diet) or vitamin A diet supplemented with BC (1500 IU vit A/kg diet +150 mg BC/kg diet) during 14 weeks.

first: Yvonne

last: van Helden

first: Erwan

last: Gouranton

first: Susanne

last: Hessel

first: Jaap

last: Keijer

first: Maria

last: Bonet

first: Johannes

last: von Lintig


title: Expression data from 9 and 13 weeks old TIFIAD1Cre mice

city: Krakow

first: Grzegorz

last: Kreiner

address: Smetna 12

department: Dept. Brain Biochemistry

country: Poland

institute: Institute of Pharmacology

summary:
We have investigated the p53-dependent stress response in medium spiny neurons (MSNs) that degenerate in Huntington’s disease. To induce p53 signaling cascade, we have genetically inactivated by the Cre/loxP system the essential RNA polymerase I (Pol I) transcription factor TIF-IA, leading to stabilization of p53 and induction of p53-dependent apoptosis.
In the present study, we selectively ablated the TIF-IA gene in MSNs by crossing TIF-IAflox/flox mice, homozygous for the floxed TIF-IA allele with transgenic mice expressing the Cre recombinase under the control of the D1 dopamine receptor (D1R) promoter.

overall_design: To explore the molecular mechanisms underlying survival and death we profiled global gene expression in 9 and 13 week old control and TIF-IAD1RCre mutant mice (3 mice/each timepoint For each of the four conditions, five GeneChips were used each )

first: Jan

last: Parkitna

first: Günther

last: Schütz

first: Rosanna

last: Parlato


title: Conversion of mouse fibroblast to hepatocyte-like cells by defined factors

status: Public on May 11 2011

city: Shanghai

first: Lijian

last: Hui

address: Yueyang Road 320

department: Institute of Biochemistry and Cell Biology

country: China

institute: Shanghai Institutes for Biological Sciences, Chinese Academy for Sciences

summary: Plasticity of differentiated cells has been proved by nuclear transfer, induced pluripotent cells and transdifferentiation. Here we show that by transduction of 3 factors (Hnf1alpha, Gata4, and Foxa3) and p19Arf inactivation, tail-tip fibroblasts can be converted to hepatocyte-like (iHep) cells, expressing hepatocyte marker genes, and acquiring many mature hepatocyte functions in vitro and in vivo.

overall_design: p19Arf-null TTFs were tranfected with 3 liver enriched transcription factors, then changed to modified Block's medium. To enrich iHep cells, epithelial cells were enriched by partial trypsin digestion.

first: Pengyu

last: Huang


title: Expression Data from Toxoplasma gondii-Infected Murine Bone Marrow-Derived Macrophages

status: Public on May 20 2011

city: Cambridge

first: Kenneth

last: Hu

state: MA

address: 31 Ames Street

department: Biology

institute: Massachusetts Institute of Technology

summary: We wanted to determine how type II Toxoplasma GRA15 and type I ROP16 affect host gene expression. We infected bone marrow-derived macrophages (BMMs) from B6 mice with type II (Pru), type II +ROP16 I, type II Δgra15, or type II Δgra15 + ROP16I.

overall_design: BMMs were plated, then infected with parasites for 18 hours. RNA was harvested from the cells by Trizol.

first: Kirk

last: Jensen

first: Jeroen

last: Saeij


title: Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice

status: Public on May 07 2011

city: Philadelphia

first: Jonathan

last: Schug

state: Pennsylvania

address: 12-156 Smilow Ctr Trans Research

department: School of Medicine

institute: University of Pennsylvania

summary: The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as CD45–/CD11b–/CD31–/MIC1-1C3+/CD133+/CD26–, at  a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creERT2-R26RYFP mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl- 3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors.

overall_design:
Treated and untreated cells were sorted by cell surface markers then compared.

M+/133+ untreated: 4 biological and 1 technical replicate - total of 5
M-/133- untreated: 2 biological and 1 technical replicate - total of 3
M+/133+ DDC-treated: 4 biological replicates - total of 4
M-/133- DDC-treated: 4 biological replicates - total of 4

first: Craig

last: Dorrell

first: Laura

last: Erker

first: Janel

last: Kopp

first: Pamela

middle: S

last: Canaday

first: Alan

last: Fox

first: Olga

last: Smirnova

first: Andrew

middle: W

last: Duncan

first: Milton

last: Finegold

first: Maike

last: Sande

first: Klaus

middle: H

last: Kaestner

first: Markus

last: Grompe


title: Deletion of astroglial Dicer causes cell non-autonomous neuronal dysfunction and degeneration

status: Public on May 10 2011

city: Los Angeles

first: Yi

last: Sun

state: CA

address: 635, Charles E. Young Dr. South

department: Molecular and Medical Pharmacology

institute: UCLA

summary: The endoribonuclease, Dicer, is indispensible for generating the majority of mature microRNAs (miRNAs), which are posttranscriptional regulators of gene expression involved in a wide range of developmental and pathological processes in mammalian central nervous system. While functions of Dicer-dependent miRNA pathways in neurons and oligodendrocytes have been extensively investigated, little is known about the role of Dicer in astrocytes. Here we report the effect of Cre-loxP mediated conditional deletion of Dicer selectively from postnatal astroglia on brain development. Dicer-deficient mice exhibited normal motor development and neurological morphology prior to postnatal week 5. Thereafter mutant mice invariably developed a rapidly fulminant neurological decline characterized by ataxia, severe progressive cerebellar degeneration, seizures, uncontrollable movements and premature death by postnatal week 9-10. Integrated transcription profiling, histological and functional analyses of cerebella showed that deletion of Dicer in cerebellar astrocytes altered the transcriptome of astrocytes to be more similar to an immature or reactive-like state prior to the onset of neurological symptoms or morphological changes. As a result, critical and mature astrocytic functions including glutamate uptake and antioxidant pathways were substantially impaired, leading to massive apoptosis of cerebellar granule cells and degeneration of Purkinje cells. Collectively, our study demonstrates the critical involvement of Dicer in normal astrocyte maturation and maintenance. Our findings also reveal non-cell autonomous roles of astrocytic Dicer-dependent pathways in regulating proper neuronal functions and implicate that loss of or dysregulation of astrocytic Dicer-dependent pathways may be involved in neurodegeneration and other neurological disorders.

overall_design: Four replicate experiments using samples derived from biologically independent pairs of control (mGfap-Cre; Dicer +/flox) and Dicer mutant (mGfap-Cre; Dicer flox/flox)  littermate mice (postnatal day 30) were performed with a dye-swap experimental design.

first: Jifang

last: Tao

first: Hao

last: Wu

middle: E


title: Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth

status: Public on May 03 2011

first: Linh

middle: My

last: Tran

state: California

department: Medicine - Pulmonary & Critical Care

institute: University of Los Angeles

summary: Alteration of the PTEN/PI3K pathway is associated with late stage and castrate resistant prostate cancer (CRPC).  However, how PTEN loss involves in CRPC development is not clear.  Here we show that castration-resistant growth is an intrinsic property of Pten-null prostate cancer (CaP) cells, independent of cancer development stage.PTEN loss suppresses androgen-responsive gene expressions by modulating androgen receptor (AR) transcription factor activity. Conditional deletion of AR in the epithelium promotes the proliferation of Pten-null cancer cells, at least in part, by down-regulating androgen-responsive gene FKBP5 and preventing PHLPP-mediated AKT inhibition. Our findings identify PI3K and AR pathway crosstalk as a mechanism of CRPC development, with potentially important implications for CaP etiology and therapy

overall_design: Mouse embryonic fibroblasts (MEFs) carrying a tet-inducible Pten transgene were generated by retro viral infection and antibiotic selection.  Cells were treated with 2 ug/ml doxycycline for 24 or 48 hours in tet-free FBS (5%)/MEF media (n=2).  Reference samples were either cells before treatment (n=2).  After each time point cells were washed twice with PBS and RNA trizol extracted.  WT samples (n =2) were also included as a control.

first: David

last: Mulholland

first: Yunfeng

last: Li

first: Houjian

last: Cai

first: Ashkan

last: Morim

first: Shunyou

last: Wang

first: Seema

middle: B

last: Plaisier

first: Isla

middle: P

last: Garraway

first: Jiaoti

last: Huang

first: Thomas

middle: G

last: Graeber

first: Hong


title: Induction of muscle-related gene expression in loose connective tissue fibroblasts in response to static tissue stretch

status: Public on May 08 2011

city: Burlington

first: Jeffrey

last: Bond

state: VT

address: 95 Carrigan Dri e

department: Microbiology and Molecular Genetics

institute: University of Vermont

summary:
Current knowledge of mechanically-induced regulation of gene expression in fibroblast is mostly based on experiments using prolonged cyclical stretching of either cultured cells or load-bearing connective tissues such as tendons and ligaments.  In this study, we have examined the effect of static tissue stretch on fibroblasts within loose connective tissue using in vivo and ex vivo models.   In an in vivo trunk side bending model, one side of the trunk of the mouse was stretched while the other side was shortened under anesthesia for 90 minutes. In the ex vivo method, whole subcutaneous tissue explants (including skin, subcutaneous muscle and subcutaneous tissue) from both sides of the trunk were incubated ex vivo either stretched or not stretched (shortened) for 24 hours.  Tissue stretch or shortening was followed by immediate dissection of the loose connective tissue layer on both sides of the trunk, RNA extraction and gene expression analysis using Affymetrix mouse gene arrays.  In both in vivo and ex vivo experiments, a large cluster of genes related to skeletal muscle function and carbohydrate metabolism was up-regulated in the stretched, compared with the shortened, connective tissue samples.  However, there were few differentially expressed matrix-related genes, and in particular no change in collagen genes.  Immunohistochemical staining for myosin after tissue stretch for 24 hours ex vivo showed increased myosin immunoreactivity in fibroblasts within the stretched compared with the shortened tissue samples. These results suggest that dynamic modulation of muscle-related gene expression is a normal physiological response of loose connective tissue fibroblasts to changes in tissue length..
Keywords: response to mechanical stretch

overall_design: Within animal stretched vs shortened (randomized): 7 animals in vivo and 4 ex vivo.

first: Helene

last: Langevin

first: Nicole

last: Bouffard

first: Gary

middle: J

last: Badger

first: Sheryl

last: White

last: Buttolph

first: Clair

last: Francomano


title: CPEB Deficiency Stimulates PTEN and Stat3 mRNA Translation and Induces Hepatic Insulin Resistance

status: Public on May 01 2011

city: Worcester

first: Mei

last: Xu

address: 55 Lake Avenue, North

department: Information service

institute: university of Massachusetts

summary: Analysis of CPEB translational regulator target mRNAs

overall_design: Microarray analysis of mRNAs associated with polysomes in wild type (WT) and Cpeb1 KO MEFs

first: Ilya

last: Alexandrov

first: Phylis

last: Patrick

first: Joel

middle: D

last: Richter


title: Foxl1-Cre-marked Adult Hepatic Progenitors Have Clonogenic and Bi-Lineage Differentiation Potential

first: Jonathan

last: Schug

summary:
The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1+ cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1+ cells had proliferative potential. Foxl1+ cells differentiated into cholangiocytes and into hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1+ cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series.

first: Soona

last: Shin

first: Gabriel

last: Walton

first: Reina

last: Aoki

first: Karrie

last: Brondell

first: Alan

last: Fox

first: Olga

last: Smirnova

first: Craig

last: Dorrell

first: Laura

last: Erker

first: Andrew

last: Chu

first: Rebecca

last: Wells

first: Markus

last: Grompe

first: Linda

last: Greenbaum

first: Klaus

last: Kaestner


title: MAP3K4/CBP Regulated H2B acetylation Controls Epithelial-Mesenchymal Transition in Trophoblast Stem Cells

status: Public on May 27 2011

city: Chapel Hill

first: Gary

last: Johnson

address: 120 Mason Farm Drive,4009 Genetic Medicine Building,CB#7365

institute: UNC-Chapel Hill

summary: Epithelial stem cells self-renew while maintaining multipotency, but the dependence of stem cell properties on maintenance of the epithelial phenotype is unclear. We previously showed that trophoblast stem (TS) cells lacking the protein kinase MAP3K4 maintain properties of both stemness and epithelial-mesenchymal transition (EMT). Here, we show that MAP3K4 controls the activity of the histone acetyltransferase CBP, and that acetylation of histone H2B by CBP is specifically required to maintain the epithelial phenotype. Combined loss of MAP3K4/CBP activity represses expression of epithelial genes and causes TS cells to undergo EMT while maintaining their self-renewal and multipotency properties. The expression profile of MAP3K4 deficient TS cells defines an H2B acetylation regulated gene signature that closely overlaps with that of human breast cancer cells. Taken together, our data define an epigenetic switch that maintains the epithelial phenotype in TS cells and reveal previously unrecognized genes potentially contributing to breast cancer.

overall_design: Three separate trophoblast stem (TS) cell conditions were compared to define the gene expression changes that occur with the induction of epithelial-mesenchymal transition (EMT) in TS cells. These conditions were TS cells differentiated for 4 days (T^Diff), TS cells differentiated for 4-days and isolated following invasion through Matrigel (T^Inv), and TS cells with an inactive MAP3K4 (TS^KI4). All conditions were normalized to wild-type control TS cells (TS^WT). T^Diff and T^Inv were analyzed in triplicate. TS^KI4 was analyzed in duplicate in two independent biological replicates.

first: Amy

middle: N

last: Abell

first: Nicole

last: Vincent Jordan

first: Weichun

last: Huang

first: Aleix

last: Prat

first: Alicia

last: Midland

first: Nancy

middle: L

first: Deborah

last: Granger

first: Piotr

last: Mieczkowski

first: Charles

last: Perou

first: Shawn

last: Gomez

first: Leping

last: Li


title: Gene expression profiling of mouse tumors arising from Keratin 6-positive mammary cells

status: Public on May 09 2011

first: Chad

last: Creighton

address: One Baylor Plaza, Mail Stop: BCM305

department: Biostatistics, Ducan Cancer Center

summary:
Conventional transgenic and knockout models do not allow selective introduction of oncogenic alterations into the progenitor population of mammary cells; thus, the role of progenitor cells in mammary tumorigenesis is yet unknown. By generating transgenic mice expressing tva – encoding the receptor for avian leukosis virus subgroup A (ALV/A) – from the Keratin 6a (K6) gene promoter,  we found that K6+ mammary cells are bipotential progenitor cells, but not stem cells.  These K6+ cells were readily induced to form tumors by intraductal injection of RCAS (an ALV/A-derived vector) carrying the gene encoding polyoma middle T antigen.  Compared with tumors induced by the same oncogene-expressing virus in transgenic mice expressing tva from the commonly used MMTV LTR or other murine models of breast cancer, tumors in this K6-tva line were unique in that they resemble the normal breast-like subtype of human breast cancer.  Consequently, these observations suggest that the cell of origin affects mammary tumor phenotypes.  This K6-tva model may be useful for preclinical testing of targeted therapy for normal-like breast cancers in patients.

Keywords: Three group comparison

overall_design: We carried out Affymetrix array analysis of five RCAS-PyMT-induced tumors each from K6-tva mice and MMTV-tva mice, as well as five mammary tumors from MMTV-PyMT transgenic mice.

first: Yi

first: Wen

last: Bu

first: Shixia


title: HDAC6 and HSP90 control the functions of Foxp3+ T regulatory cells

first: Wayne

last: Hancock

state: PA

address: 916B ARC, 3615 Civic Ctr Blvd

institute: CHOP/UPenn

summary: Foxp3+ T-regulatory cells (Tregs) are key to immune homeostasis such that their diminished numbers or function can cause autoimmunity and allograft rejection. Foxp3+ Tregs express histone/protein deacetylases (HDACs) that regulate chromatin remodeling, gene expression and protein function. Pan-HDAC inhibitors developed for oncology enhance Treg production and suppression but have limited non-oncologic applications given their broad effects. We show, using HDAC6-deficient mice and WT mice treated with HDAC6-specific inhibitors, that HDAC6 inhibition promotes Treg suppressive activity in models of inflammation and autoimmunity, including multiple forms of experimental colitis and fully MHC-incompatible cardiac allograft rejection. Many of the beneficial effects of HDAC6 targeting are also achieved by inhibition of the HDAC6-regulated protein, HSP90. Hence, selective targeting of a single HDAC isoform, HDAC6, or its downstream target, HSP90, can promote Treg-dependent suppression of autoimmunity and transplant rejection.

overall_design: RNA from three independent samples from magnetically separated CD4+CD25+ Treg of HDAC6 knock out, compared to wild type (C57BL6) control

first: Edwin

middle: F

last: de Zoeten

first: Liqing

last: Wang

first: Kyle

last: Butler

first: Ulf

last: Beier

first: Hong

last: Sai

first: James

last: Bradner

first: Ralph

last: Mazitschek

first: Alan

last: Kozikowski

first: Patrick

last: Matthias


title: Foxl1-Cre-marked Adult Hepatic Progenitors Have Clonogenic and Bi-Lineage Differentiation Potential - Time Course

first: Jonathan

last: Schug

summary: The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1+ cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1+ cells had proliferative potential. Foxl1+ cells differentiated into cholangiocytes and into hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1+ cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

overall_design: There were 26 samples used in the analysis.  Typically 4 replicates for each of 7 conditions, except for two condtions which had only 3 replicates.  Day 0 samples are non-parenchymal cells without DDC treatment.  YFP_pos samples are YFP-expressing non-parenchymal cells induced by Foxl1-CRE after 3, 7 or 14 days of DDC treatment.  YFP_neg samples are analogous but do not expressed YFP.

first: Soona

last: Shin

first: Gabriel

last: Walton

first: Reina

last: Aoki

first: Karrie

last: Brondell

last: Fox

first: Olga

last: Smirnova

first: Craig

last: Dorrell

first: Laura

last: Erker

first: Andrew

last: Chu

first: Rebecca

last: Wells

first: Markus

last: Grompe

first: Linda

last: Greenbaum

first: Klaus

last: Kaestner


title: Wild type N-ras displays anti-malignant properties, in part by downregulating decorin

status: Public on Apr 19 2011

city: LYON

first: Jiri

last: ZAVADIL

state: Other

address: 150 COURS ALBERT THOMAS

country: France

institute: IARC WHO

summary:
Previously, we have demonstrated that wild type N-ras (wt N-ras) harbors an anti-malignant effect against mutated Ras and in tumors without Ras mutations. To investigate the molecular bases of this anti-malignant activity, we have studied the potency of this anti-malignant effect in a model system against SV40 T. We show that wild-type N-ras (wt N-ras) counteracts the effects of SV40T in NIH3T3 cells as seen by a decrease in proliferation, anchorage independence and changes in migration). We also show that wt N-ras also elicits the same anti-malignant effects in some human tumor cell lines (HT1080 and MDA-MB-231). Through mRNA and microRNA (miRNAs) expression profiling we have identified genes and miRNAs modulated by wt N-ras potentially responsible for the anti-malignant effect. wt N-ras appears to mediate its anti-malignant effect by downregulating some of the targets of the TGFb pathway and decorin, which are able to reverse the inhibition of migration induced by wt N-ras. Our experiments show that the molecules that mediate the anti-malignant effect appear to be different than those modulated by transforming N-ras. The components of the pathways modulated by wt N-ras mediating its anti-malignant effects are potential targets for therapeutic intervention in cancer.
We report that the N-ras proto-oncogene has anti-malignant effects and that it does so by modulating different genes than its oncogenic counterpart, which indicates that it should be activating different pathways.

overall_design: Gene expression profiles from 3T3 cells transformed by SV40 (SV5) and cells expressing both SV40T and wt N-ras (SV5-NN clones) were compared using genome wide mRNA expression profiling by Affymetrix genechip arrays (Mouse 430 2.0) and key targets were validated by real time RT-PCR.

first: Marta

last: Benet

first: Robin

last: Yates-Dulman

first: Raffi

last: Suzme

first: Eleazar

last: Vega-Saenz de Miera

first: Martha

last: Vega

first: Thuy

last: Nguyen

last: Zavadil

first: Angel

last: Pellicer


title: Gene expression data from teratomas formed by C57BL/6 (B6) ES and EiPS cells in B6 mice

status: Public on Apr 13 2011

city: La Jolla

first: Tongbiao

last: Zhao

address: 9500 Gilman Dr

department: Biological Sciences

institute: University of California San Diego

summary:
Embryonic fibroblast from C57BL/6 (B6) mice were reprogrammed to EiPS without exogenous DNA integration using an single episomal vector. The EiPS cells and B6 ES cells were then transplanted into B6 mice to form teratomas.
We used microarrays to profile the gene expression differences between the teratomas formed by B6 ES cells and EiPS cells.

overall_design: Pooled RNA from two teratomas formed by B6 ES or two regression teratomas formed by EiPS were selected for hybridization on Affymetrix microarrays.

first: Yang

last: Xu


title: A signature of 6 genes highlights defects on cell growth and specific metabolic pathways in murine and human hepatocellular carcinoma.

status: Public on Apr 23 2011

city: Pamplona

first: Paul

middle: C.

last: Schröder

email: pschrode@unav.es, fjcorrales@unav.es

state: Navarra

address: Avda. Pío XII, 55

department: Gene Therapy & Hepatology

country: Spain

institute: CIMA (Center for Applied Medical Research) University of Navarra

summary: Hepatocellular carcinoma (HCC) represents a major health problem as it afflicts an increasing number of patients worldwide. Albeit most of the risk factors for HCC are known, this is a deadly syndrome with a life expectancy at the time of diagnosis of less than 1 year. Definition of the molecular principles governing the neoplastic transformation of the liver is an urgent need to facilitate the clinical management of patients, based on innovative methods to detect the disease in its early stages and on more efficient therapies. In the present study we have combined the analysis of a murine model and human samples of HCC to identify genes differentially expressed early in the process of hepatocarcinogenesis, using a microarray based approach. Expression of 190 genes was impaired in murine HCC from which 65 were further validated by low-density array RT PCR.  The expression of the best 45 genes was then investigated in human samples resulting in 18 genes which expression was significantly modified in HCC. Among them, JUN, methionine adenosyltransferase 1A and 2A, phosphoglucomutase 1, and acyl CoA dehydrogenase short branched chain indicate defective cell proliferation as well as one carbon pathway, glucose and fatty acid metabolism, both in HCC and cirrhotic liver, a well known preneoplastic condition. These alterations were further confirmed in public transcriptomic datasets from other authors. In addition, vasodilator stimulated phosphoprotein, an actin-associated protein involved in cytoskeleton remodelling, was also found to be increased in the liver and serum of cirrhotic and HCC patients. In addition to revealing the impairment of central metabolic pathways for liver homeostasis, further studies may probe the potential value of the reported genes for the early detection of HCC.

overall_design: Transcriptomic analysis of tumors from MAT1A deficient mice

middle: C

first: Víctor

last: Segura

first: José

middle: I

last: Riezu

first: Bruno

last: Sangro

last: Mato

first: Jesús

last: Prieto

first: Enrique

last: Santamaría

first: Fernando

last: Corrales


title: ApoD modulation mouse cerebellar transcriptome

status: Public on Apr 16 2011

city: Sevilla

first: Gabriel

last: Gutierrez

address: PO BOX 1095

department: Genetics

institute: University of Seville

summary: The lipocalin Apolipoprotein D (ApoD), known to protect the nervous system against oxidative stress (OS) in model organisms, is up-regulated early in the mouse brain in response to the ROS generator paraquat (PQ). However, the processes triggered by this up-regulation have not been explored.

overall_design: We present here a study of the effect of ApoD on the early transcriptional changes upon OS in the mouse cerebellum using microarray profiling. ApoD-KO and transgenic mice over-expressing ApoD in neurons are compared to wild-type controls.

first: Diego

last: Sánchez

last: Gutiérrez


title: Comparative global gene expression profiling of mice liver and spleen fed with nucleoprotein (NP) diets

city: Tsukuba

first: RANDEEP

last: RAKWAL

state: Ibaraki

address: 1-1-1 Tennodai

department: Institute of Health and Sport Sciences

institute: University of Tsukuba

summary: A diet rich in nucleic acids and protamin protein, termed as nucleoprotein was used for the study. Mice were fed with NP diets for 4 weeks followed by removal of the liver and spleen. Total RNA extracted from livers and spleens was pooled in each group (low NP or LNP-control, and 1.2% NP-treatment, diets), prior to DNA microarray analysis (Agilent mouse whole genome 4 x 44K). Results revealed 1373 & 3386 up (>1.5 fold)- and down (<0.75 fold)-regulated genes in the liver, and 252 & 1838 up- and down-regulated genes in the spleen, respectively following 1.2%NP diet. Analysis of genes related to NP diets will be discussed.

overall_design: To investigate the effect of NP, we added S-nuclegen® at a concentration of 1.2% into CLEA basic purified diet (CLEA JAPAN, Inc., Tokyo, Japan) known to include nucleic acids (NAs) at much lower amounts than standard diet. By HPLC analysis CLEA basic purified diet (low NP), and 1.2% NP diet included 0.03%, and 0.5% NAs, respectively. Male C57BL/6J mice (13 weeks) were fed with NP diet for 4 weeks. The liver and spleen were removed and deep frozen in liquid nitrogen. Whole blood was also sampled from these mice, and frozen in liquid nitrogen. Total RNA extracted from livers and spleens was pooled in each group (control, lowNP or LNP; and treatment, 1.2%NP), prior to DNA microarray analysis (Agilent mouse whole genome 4 x 44K).

first: Tetsuo

last: Ogawa

first: Tomomi

last: Saito

first: Chika

last: Sawa

first: Aya

last: Murayama

first: Randeep

last: Rakwal

first: Junko

last: Shibato

first: Seiji

last: Shioda


title: SIRT1 impact on global gene expression in the brain

status: Public on Apr 22 2011

first: Sergiy

last: Libert

address: 77 Mass ave

institute: MIT

summary: We compared expression of genes in brains of SIRT1 brain-specific knockouts (BSKO) to those of wild-type littermate controls (WT).

overall_design: RNA was isolated from whole brain tissues of SIRT1 brain specific knockout mice (BSKO) and their wild-type littermates (WT). 3 months old males were used, four biological replicates per genotype were used. RNA was verified for integrity and processed according to Affymetrix guidelines, using kits and reagents supplied by Affymetrix. Total of 8 Affymetrix.GeneChip.Mouse430A_2 chips single channel were used.

first: Leonard

last: Guarente


title: Alteration of gene expression patterns in the liver and spleen following exposure to nucleoprotein diet in the mouse LPS injection model

status: Public on Apr 01 2011

summary: A diet rich in nucleic acids and protamin protein, termed as nucleoprotein (NP), has been attracting a great deal of attention in food science for its beneficial effects. In the present study, we performed a global gene expression profiling in mice fed with NP diet rich in nucleoproteins from the salmon testis. Since our recent research has revealed anti-inflammatory effect of the NP diet, we induced inflammation by lipopolysaccharide (LPS) injection in the mice for this analysis study. Mice were fed with NP diet for 4 weeks followed by a single injection of LPS. The liver and spleen were removed 6 h post-LPS injection. Total RNA extracted from livers (L) and spleens (S) was pooled in each group (control and NP diet), prior to DNA microarray analysis (Agilent mouse whole genome 4 x 44K). Results revealed 322 & 702 up (>1.5 fold)- and down (<0.75 fold)-regulated genes in the liver, and 325 & 501 up- and down-regulated genes in the spleen, respectively following NP diet. Analysis of genes related to inflammation suggests increased activity of immune function during acute period of LPS injection, which may contribute to early demise of inflammation. NP diet can be expected to be useful for inhibition of inflammatory reactions whose over-accumulation is thought to be related to the acceleration of aging process.

overall_design: To investigate the effect of NP, we added S-nuclegen® at a concentration of 1.2% into CLEA basic purified diet (CLEA JAPAN, Inc., Tokyo, Japan) known to include nucleic acids (NAs) at much lower amounts than standard diet. By HPLC analysis CLEA basic purified diet (low NP), and 1.2% NP diet included 0.03%, and 0.5% NAs, respectively. Male C57BL/6J mice (7 weeks) were fed with NP diet for 4 weeks followed by a single injection of LPS at a dose of 10 mg/kg. The liver and spleen were removed 6 h post-LPS injection. Total RNA extracted from livers and spleens was pooled in each group (control and NP diet), prior to DNA microarray analysis (Agilent mouse whole genome 4 x 44K).


title: Complex modulation of androgen responsive gene expression by methoxyacetic acid

status: Public on Apr 07 2011

city: Boston

first: David

middle: J.

last: Waxman

address: 5 Cummington Mall

department: Department of Biology and Bioinformatics Program

institute: Boston University

summary: Optimal androgen signaling is critical for testicular development and spermatogenesis.  Methoxyacetic acid (MAA), the primary active metabolite of the industrial chemical ethylene glycol monomethyl ether, disrupts spermatogenesis and causes testicular atrophy.  Transcriptional trans-activation studies have indicated that MAA can enhance androgen receptor activity, however, whether MAA actually impacts the expression of androgen-responsive genes in vivo, and which genes might be affected is not known.  A mouse TM3 Leydig cell line that stably expresses androgen receptor (TM3-AR) was prepared and analyzed by transcriptional profiling to identify target gene interactions between MAA and testosterone on a global scale.  MAA is shown to have widespread effects on androgen-responsive genes, affecting processes ranging from apoptosis to ion transport, cell adhesion, phosphorylation and transcription, with MAA able to enhance, as well as antagonize, androgenic responses. Moreover, testosterone is shown to exert both positive and negative effects on MAA gene responses.  Motif analysis indicated that binding sites for FOX, HOX, LEF/TCF, STAT5 and MEF2 family transcription factors are among the most highly enriched in genes regulated by testosterone and MAA. Notably, 65 FOXO targets were repressed by testosterone or showed repression enhanced by MAA when combined with testosterone; these include 16 genes associated with developmental processes, six of which are Hox genes.  These findings highlight the complex interactions between testosterone and MAA, and provide insight into the effects of MAA exposure on androgen-dependent processes in a Leydig cell model.

overall_design: TM3-AR cells (see below) were grown in DMEM-F12 medium containing 5% horse serum and 2.5% FBS.  LNCaP cells were maintained in RPMI 1640 containing 10% FBS.  RNA was isolated using TRIzol reagent using the manufacturer’s protocol.


title: Developmental influence of the cellular prion protein on the gene expression profile in mouse hippocampus

status: Public on Apr 12 2011

city: Trieste

first: Paola

last: Roncaglia

state: TS

address: via Bonomea 265

department: Neurobiology

country: Italy

institute: SISSA/ISAS (International School for Advanced Studies)

summary:
A key event in the pathogenic process of prion diseases is the conversion of the cellular prion protein (PrPC) to an abnormal and protease-resistant isoform (PrPSc). Mice lacking PrP are resistant to prion infection, and down-regulation of PrPC during prion infection prevents neuronal loss and the progression to clinical disease. These results are suggestive of the potential beneficial effect of silencing PrPC during prion diseases. However, the silencing of a protein that is widely expressed throughout the CNS could be detrimental to brain homeostasis. The physiological role of PrPC remains still unclear, but several putative functions have been proposed. Among these, several lines of evidence support PrPC function in neuronal development and maintenance.
To assess the influence of PrPC on gene expression profile during development in the mouse brain, we undertook a microarray analysis by using RNA isolated from the hippocampus, at two different developmental stages: newborn (4-day-old) and adult (3-month-old) mice, both from Prnp+/+ and Prnp0/0 animals. The comparison of the different datasets allowed us to identify “commonly” co-regulated genes and “uniquely” de-regulated genes during postnatal development in these animal models.  The lack of PrPC during neuronal development affected several biological pathways, among which the most representative were cell signaling, cell-cell communication and transduction process. In addition, the absence of PrPC influenced genes involved in calcium homeostasis, nervous system development, synaptic transmission and cell adhesion. There was only a moderate alteration of the gene expression profile during neuronal development in the animal models we studied. PrPC deficiency does not lead to a dramatic alteration of gene expression profile, and produces moderate altered gene expression levels from young to adult animals. Thus, our results may provide additional support to silencing endogenous PrPC levels as a therapeutic approach to prion diseases.

overall_design: To analyze the influence of PrPC expression on CNS gene expression profile during development, we investigated WT PrPC (Prnp+/+) and Prnp0/0 mice at two different developmental stages: in neonatal animals (postnatal day 4, P4) and in adult animals (3 months old). For each developmental stage, hippocampi of 3 (pups) or 4 (adult) animals were dissected immediately after animal sacrifice and promptly processed for RNA extraction and purification, for a total of 14 samples.


title: Seeking Gene Candidates Responsible for Developmental Origins of Health and Disease (DOHaD)

summary: Human epidemiological evidence has led scientists to theorize that undernutrition during gestation is an important early origin of adult diseases. Animal models have successfully demonstrated that maternal diet could contribute to some of adult diseases. Undernutrition is perceived harmful in pregnant women whereas calorie restriction is a strategy proven to extend healthy and maximum life span in adult. This diagrammatically opposite effect of nutritional condition might provide us hints to search for genes underlying health conditions. Here, we have initiated a study examining the effect of undernutrition on maternal and fetal livers, utilizing high-throughput DNA microarray analysis for screening genome-wide changes in their transcriptomes. Briefly, pregnant mice were exposed to food deprivation (FD) on gestation day (GD) 17, and caesarean section was performed on GD18. Control mice were supplied with chow ad libitum till sacrifice. Total RNA extracted from mother and fetal livers for each control and treatment (FD) was analyzed with an Agilent mouse whole genome DNA chip. Total of 3058 and 3126 up (>1.5 fold)- and down (<0.75 fold)-regulated genes, and 1475 and 1225 up- (>1.5 fold)- and down (<0.75 fold)-regulated genes showed differential expression at the mRNA level, in the maternal and fetal livers, respectively. Interestingly, 103 genes up-regulated in mother were down-regulated in fetus, whereas 108 down-regulated maternal genes were up-regulated in fetus; these 211 genes are potential candidates related to longevity or health. The role of some of these genes, in context of the proposed mechanisms for developmental origins of health and disease is discussed.

overall_design: Comparison between mouse control and FD livers in fetus and mother was performed. Five to ten biological replicates were used, and pooled total RNA from each condition (control and FD) was dye-swaped.

first: Makiko

last: Kuwagata

first: Haruaki

last: Kageyama

first: Michiko

last: Yagi

first: Kazue

last: Satoh


title: Mouse embryo heart, e10.5 AVC: Control vs. Tbx3 hypomorphic mutant

city: Salt Lake City

first: Deborah

last: Frank

state: UT

address: 15 North 2030 East

department: Pediatrics

institute: University of Utah

summary:
Transcriptional profiling of the atrioventricular canal from e10.5 mouse hearts comparing wild-type control with Tbx3 GH/N hypomorphic mutant microdissected tissue.
The goal was to identify direct and indirect targets of the transcription factor Tbx3, because Tbx3 is expressed in the AVC and hypomorphs develop embryonic AV conduction block at e12.5.

overall_design: Two-condition experiment, wild-type control and Tbx3 mutant AVC tissue.  Tissue from 5 hearts was pooled to make each sample.  Biological replicates: 4.

middle: U

first: Anne

middle: M

last: Moon


title: Adipocyte differentiation with or without Bilberry and anthocyanidins

city: Sinjuku-ku

first: Masami

last: Tanaka

state: Tokyo

address: Sinjuku 6-1-1

institute: Tokyo Medical University

summary: Metabolic syndrome is an important public concern and demand for effective therapeutic strategies. Abdominal obesity, especially an increase in the visceral adipose tissue, is the main cause of this syndrome. Flavonoids are expected to improve risk factors for metabolic syndrome. Bilberry, original species of blueberry containing anthocyanidin flavonoids have been used for centuries in Europe to ameliorate the symptoms of diabetes, but their effects and the mechanisms on lipid accumulation of adipocyte cells are not well defined. In the present study, we investigated effects of the Bilberry extract on differentiation of adipocytes using 3T3-L1 adipocyte cell line. Exposure to Bilberry extract during the early period of adipogenesis (6 days) was significantly inhibited adipocyte differentiation of 3T3-L1. During this period, Bilberry extract greatly down-regulated the mRNA levels of the key adipogenesis-associated markers peroxisome proliferator-activated receptor-γ (PPARγ). Furthermore, Bilberry extract significantly decreased expression of the transcription factor Sterol Regulatory Element Binding Protein 1c (SREBP1c), which plays a central role in adipocyte differentiation including the induction of PPARγ. The expression of SREBP1c is remarkably enhanced in response to insulin, thus raises the possibility that Bilberry extract might inhibit the Insulin pathway. So, We investigated whether Billberry extract and anthocyanidines turned the insulin signaling pathway using microarray. As a result, Gene Set Enrichment Analysis (GSEA) shows that these additives turned insulin signaling pathway certainly.

overall_design: We quantified expression profiles of whole mRNAs by microarray in the adipocyte from 3T3-L1 with or without additives. 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA) were grown to confluence in DMEM media (Sigma Aldrich, Tokyo, Japan) with 10% calf serum and penicillin (100 U/ml)/streptomycin (100 ug/ml). Adipogenesis was induced using an adipogenesis assay kit (Chemicon International, Temecula, CA). On day 0, cells were induced with initiation media (10 ug/ml insulin, 1 uM dexamethasone and 0.5 uM IBMX in DMEM media added with 1: 0.1% DMSO, 2: 100 ug/ml Bilberry extract, 3: 100 nM Delphinidin or 4: 100 nM Cyanidin (total 4 samples). On day 2, harvested the cells, extracted total RNA and did microarray experiments.

first: Rieko

last: Suzuki

first: Masakatsu

last: Takanashi

first: Hiroo

last: Toyoda

first: Masahiko

last: Kuroda


title: Expression data from lateral nasal gland of male B6, 129/Sv and Cyp2g1-null mice

city: Albany

first: Xin

last: Zhou

state: NY

address: Empire State Plaza P.O. Box 509

department: NY State Dept. of Health

institute: Wadsworth Center

summary: In male Cyp2g1-null mice, the lateral nasal gland, one of the largest anterior glands in the nasal cavity, was found to be protected from acetaminophen toxicity. The goal of this study was to identify the genes that are involved in the mechanisms, especially those genes functional in drug metabolism, clearance and post-activation events.

overall_design: Lateral nasal gland from 2-3-month-old male mice were dissected from each strain of mouse (B6, 129/Sv and Cyp2g1-null). Both B6 and 129/Sv are used as control mice because the Cyp2g1-null mice are on the mixed genetic background. Glands from 10 mice of each strain were pooled for RNA preparaion for one chip. There are totally 9 chips including 2 for 129/Sv, four for B6 and 3 for Cyp2g1-null mice, respectively.


title: Leukemias of different origins

status: Public on Mar 31 2011

first: Andrei

middle: V.

last: Krivtsov

address: 1 Blackfan circle

department: Hem Onc

institute: Children's Hospital

summary: We generated MLL-AF9 mediated murine leukemias that originate either from hematopoietic stem or committed progenitors cells. The luekemia stem cell fraction in these two type of leukemias shared exactly the same immunophenotype but their genetic programs differ.

overall_design: Total RNA from HSC(KLS), CMP, MEP, and GMP, and from leukemia stem cells (LGMP)  was isolated and hybridized to Affymetrix expresison microarrays.

middle: V

first: Scott

middle: A

last: Armstrong


title: Nuclear receptor, RORa plays a critical role in obesity-associated inflammation, hepatic steatosis, and insulin resistance

city: RTP

first: NIEHS

last: Microarray Core

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

summary: We demonstrated that RORa-deficient staggerer mice (RORasg/sg) fed with a high fat diet (HFD) showed reduced adiposity and hepatic triglyceride levels compared to wild type (WT) littermates and were resistant to the development of hepatic steatosis, adipose-associated inflammation, and insulin resistance. Gene expression profiling showed that many genes involved in triglyceride synthesis and storage, including Cidec, Cidea, and Mogat1, were expressed at much lower levels in liver of RORasg/sg mice. In addition to reduced lipid accumulation, inflammation was greatly diminished in white adipose tissue (WAT) of RORasg/sg mice fed with a HFD. The infiltration of macrophages and the expression of many immune-response and pro-inflammatory genes, including those encoding various chemo/cytokines, toll-like receptors, and TNF signaling proteins, were significantly reduced in RORasg/sg WAT. Moreover, RORasg/sg mice fed with a HFD were protected from the development of insulin resistance. Together, these results indicate that RORa plays a critical role in the regulation of several aspects of metabolic syndrome. Therefore, RORa may provide a novel therapeutic target in the management of obesity and associated metabolic diseases.

overall_design: Liver and white adipose tissue (WAT) total RNAs were purified from 5 WT and 5 RORasg/sg (natural deletion of RORa gene in mice) mice fed with a high fat diet for 6 weeks. Then samples were applied on Agilent mouse genome chip.

first: HongSoon

last: Kang

first: Kyoko

last: Okamoto

first: Anton

last: Jetten


title: The Involvement of mir-146 in Microglial Cells (EOC 13.31) During Prion Disease

status: Public on Jul 01 2011

city: Winnipeg

first: Rhiannon

middle: LCH

last: Huzarewich

state: MB

address: 1015 Arlington St.

department: Molecular PathoBiology

institute: Public Health Agency of Canada

summary: MicroRNAs (miRNAs) are evolutionary conserved, non-coding, gene regulatory RNA molecules found in both plants and animals and amongst almost every cell and tissue type. They are about 22 nucleotides long and are involved in silencing of mRNA through sequence specific binding to the 3’ untranslated region (UTR) of the mRNA subsequently causing translational repression and/or will promote the degradation of protein-coding mRNA. Specifically, the miRNA family, mir-146 a/b, has been previously found to be involved in the regulation of the innate immune response by functioning as a negative regulator to help fine-tune the immune response.   Microglial cells are the macrophage of the brain participating as major players of the innate immune response. During prion disease, no immune response is mounted against PrPSc possibly due to its similarity to host PrPc and thus, the host immune response would be suppressed and tightly regulated. Therefore, an increased expression of mir-146 by microglial cells during prion disease may function as one of these negative regulators.      Our objective of this experiment is to use DNA microarrays to investigate the gene regulation of mir-146 previously found upregulated in our studies of mouse brain tissue specifically in microglial cells during prion disease with the aim of having a better understanding of prion pathobiology and a potential target for therapeutic intervention.

overall_design: Mir-146 expression was confirmed via in situ analysis of brain tissue and was further investigated in a microglial murine cell line (EOC 13.31). To mimic mir-146 upregulation during prion disease, mir-146 a & b were overexpressed in EOCs using a lipid based reverse transfection system. Furthermore, endogeneous mir-146 was knocked down as another method to help confirm the mRNA targets affected by mir-146. Simultaneously, another experiment was performed for the investigation of its involvement in the innate immune response by stimulating the EOCs with differing concentrations of LPS from E.coli (055:B5). Total RNA was collected and prepared at several timepoints and the levels of expression of both mir-146 a & b was tested via qRT-PCR. The RNA collected from the EOCs from each condition were used as target material for dual-color, competitive hybridization to Agilent whole mouse genome 4x44K oligo arrays. All the significant targets found on the mircorarrays were compared against the various conditions to find a consensus of affected mir-146 mRNA targets.

first: Stephanie

last: Booth


title: Expression data from Jejunums of C57BL/6J mice

status: Public on Mar 20 2011

city: Wuxi

first: Guo-wei

last: Le

address: Lihu Road 1800

institute: Jiangnan University

summary:
Many diseases in digestive system have been improved related to the uptake of high-carbohydrate diet. The underlying mechanism might be related the changes of genes expression in digestive system.
We used microarrays to detail the global programme of gene expression  during this process.

overall_design: Jejunums from mice fed with high-glucose diet or control were selected  for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain populations of genes related to lipid metabolism in order to increase the temporal resolution of expression profiles. To that end, we analyzed the expressions of genes related to lipid metabolism according to clustering analysis and references.

first: Dan

last: Du

first: Yong-Hui

last: Shi

first: Guo-Wei


title: Rhabdomyosarcoma can be initiated in activated Pax7+ Muscle Progenitor Cells

status: Public on Jan 30 2011

city: Durham

first: Jeffrey

last: Mito

institute: Duke University Medical Center

summary: Microarray analysis of rhabdomyosarcomas generated using mice with conditional mutations in Kras and p53 using both in vivo and in vitro approaches to identify a cell of origin for rhabdomyosarcoma.

overall_design: Contains rhabdomyosarcomas derived in vivo using Pax7-CreER mice and in vitro using sorted muscle progenitor cells, UPS derived from intramuscular Adeno-Cre injection, and normal muscle.

first: Jordan

last: Blum

first: David

last: Kirsch


title: KLF2-Regulated Gene Expression in Mouse Embryonic Yolk Sac Erythroid Cells

status: Public on Mar 02 2011

city: Richmond

first: Latasha

last: Redmond

state: Virginia

address: 401 College Street

department: Department of Human and Molecular Genetics

institute: Virginia Commonwealth University

summary: KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell, and erythroid development. KLF2-/- mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. Embryonic -like globin gene expression is reduced in KLF2-/- embryos compared to wildtype (WT), and E10.5 erythroid cells exhibit abnormal morphology. Other KLF2 target genes were identified by comparing E9.5 KLF2-/- and WT yolk sac erythroid cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression; eighty-nine of these are downregulated in KLF2-/- compared to WT. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. Previously identified erythroid-enriched regulatory genes such as reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2-/- compared to WT. SOX2, a pluripotency factor in ES cells, is also a KLF2 target in embryonic erythroid cells. We investigated whether reelin, which has an established role in neuronal migration and proliferation, has a role in embryonic erythropoiesis. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter, but reelin mutant mice have no apparent abnormalities in embryonic erythroid morphology or globin gene expression.

overall_design: Timed-pregnant KLF2+/- females were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo,  cryoprotected in 20% sucrose in PBS and  frozen in OCT media.   A small portion of the embryo tail was used for PCR genotyping. Eight micron KLF2-/-  frozen yolk sac sections were obtained and  laser capture microdissection (LCM) was used to isolate primitive erythroid precursors. For each biological replicate, 2 to 4 yolk sacs from 2 different litters were used. Total RNA was isolated from 4 different KLF2-/-  erythroid samples and hybridized to Affymetrix 430 A 2.0 microarrays

first: Catherine

last: Dumur

first: Kellie

last: Archer

first: Dennis

middle: R

last: Grayson

first: Jack

last: Haar

first: Joyce

last: Lloyd


title: Comparison of Gene expression pattern between heat-killed M.leprae and live M. leprae-infected peritoneal macrophage in vivo model

status: Public on Mar 17 2011

city: Seoul

first: Tae Hoon

last: Kim

state: Socho-gu

address: 505 Bonpo-dong

department: Pathology

country: South Korea

institute: Medical College of the Catholic University

summary: Our goal is to discriminate specific genes in live M.leprae-infected peritoneal macrophages in comparison to heat-killed M.leprae infected peritoneal macrophages using microarray.

overall_design: Two-condition experiment, Heat-killed M.leprae infected macrophage vs. Live M.leprae infected macrophage. Biological replicates: 16 mice (control), 8 heat-killed M. leprae mice (sample 1), and 8 live M. leprae infected mice (sample 2). Independently grown and harvested from isolator. One replicate per array.

first: Tae

middle: H

first: Su

middle: Y

first: Hyo

last: Eun

last: Kang

first: Hye

middle: W

last: Park

first: Seung

first: Gue

middle: T

last: Chae


title: Isolation of epiblast stem cells from pre-implantation mouse embryos

status: Public on Jan 25 2011

city: Cleveland

first: Paul

last: Tesar

state: Ohio

address: 10900 Euclid Ave.

department: Genetics & Genome Sciences

institute: Case Western Reserve University

summary: Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined.  We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively.  Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos.  Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos.  The pre-implantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs.  These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.

overall_design: 6 total samples were analyzed.  Three pluripotent cell types (mES cells, E3.5 EpiSCs, and E5.5 EpiSCs) were compared with and without treatment of SB431542 for 4 days.


title: E47 KO versus WT HSCs

status: Public on Jan 22 2011

city: Drexel Hill

first: Qi

last: Yang

address: 82 Ferne Blvd, Apt6

institute: University of Pennsylvania

summary: Genome-wide gene expression pattern of E47 KO versus WT HSCs from primary and secondary recipient mice were analysis using Agilent one-color micro-array analysis.

overall_design: Two replicates from 4 samples are analyzed. The sample from the WT mouse were set as their reference sample.

first: Lisa

last: Borghesi

first: Jana

last: Ciomperlik

first: Bernhard

last: Gerstmayer


title: Signatures of murine B-cell development implicate Yy1 as a regulator of the germinal center-specific program

status: Public on Feb 14 2011

first: Michael

middle: Richard

last: Green

state: MA

address: 44 Binney Street

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary:
Heirarchical development of B-cells involves the induction and supression of large sets of genes that provide the basis for differentiation and, ultimately, antibody production.
We used microarrays to define comprehensive differentiation state-specific transcriptional signatures in order to gain insight into B-cell biology. In addition, we compared profiles of normal B-cells to those of murine lymphoma models that are putatively aligned with different states.

overall_design: B-cells of different differentiation states were purified from the bone marrow and spleen of mice use flow cytometry with antibodies specific for pre-defined markers of each differentiation state. Purified populations were then profiled using Affymetrix Mouse 430A2 gene expression microarrays. Tumors from Bcl6-trangenic mice, Bcl6/Myc dual-transgenic mice and p53/Lig4 double knock-out mice were also profiled using these microarrays in order to assess the similarities/differences between normal and malignant B-cells.

first: John

last: Manis


title: Polidocanol injury wildtype versus Myd88 KO trachea

city: London

first: Adam

last: Giangreco

address: 5 University Street

department: Respiratory Research

country: United Kingdom

institute: UCL

summary: Nf-kB activity is associated with the key pathological features of chronic respiratory diseases including epithelial remodelling, excess mucous production, and submucosal gland hyperplasia.  However, the role of Nf-kB activity in airway epithelial differentiation remains controversial.  In the present study we demonstrate that Nf-kB adaptor protein Myd88 deficiency promotes increased airway submucosal gland abundance and abnormal epithelial differentiation in proximal adult airways.  Abnormal airway differentiation was not developmentally determined, became exacerbated following acute lung injury, and did not involve altered epithelial proliferation or apoptosis.  Instead, we demonstrate that tracheal Myd88 deficiency promotes upregulation of a unique gene expression profile that includes activation of alternate, Myd88-independent Nf-kB signalling.  Finally, we show that these effects are not intrinsically maintained in vitro using an air-liquid interface epithelial culture.  This finding indicates that Myd88 deficiency promotes adult airway remodelling by regulating non-epithelial, non-cell autonomous Nf-kB activity.

overall_design:
20 microarray samples of whole trachea RNA in total:
5 samples wildtype control tissue
5 samples Myd88 KO control tissue
5 samples wildtype 3 day polidocanol injury tissue
5 samples Myd88 KO 3 day polidocanol injury tissue

first: Liwen

last: Lu

first: Dawn

middle: J

last: Mazzatti

first: Bradley

last: Spencer-Dene

first: Sam

last: Janes


title: Analysis of the host microRNA response to Salmonella uncovers the control of major cytokines by the let-7 family

status: Public on Mar 10 2011

city: Berlin

first: Hans-Joachim

last: Mollenkopf

address: Charitéplatz 1

institute: Max-Planck-Institute for Infection Biology

summary:
MicroRNAs have well-established roles in eukaryotic host responses to viruses and extracellular bacterial pathogens. In contrast, microRNA responses to invasive bacteria have remained unknown. Here, we report cell type-dependent microRNA regulations upon infection of mammalian cells with the enteroinvasive pathogen, Salmonella Typhimurium. Murine macrophages strongly up-regulate NF-κB associated microRNAs; strikingly, these regulations which are induced by the bacterial lipopolysaccharides (LPS) occur and persist regardless of successful host invasion and/or replication, or whether an inflammatory response is mounted, suggesting that microRNAs belong to the first line of anti-bacterial defence. However, a suppression of the global immune regulator miR-155 in endotoxintolerant
macrophages revealed that microRNA responses also depend on the status of infected cells. This study identifies the let-7 family as the common denominator of Salmonella regulated microRNAs in macrophages and epithelial cells, and suggests that repression of let-7 relieves cytokine IL-6 and IL-10 mRNAs from negative post-transcriptional control. Our results establish a paradigm of microRNA-mediated feed-forward activation of inflammatory factors when mammalian cells are targeted by bacterial pathogens.

overall_design:
The murine macrophage cell-line RAW 264.7 was exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication.
We compared mouse mRNA abundances between cells infected with wild-type ("24WT") or SPI1/2 knockout Salmonella ("24SPI1/2") and non-infected, mock-treated controls ("24C"). Both strains mounted a similar inflammatory response as judged from the abundance of pro-inflammatory genes compared to the controls. These data indicate, that macrophages can mount a full inflammatory response to extracellular bacteria, that is not further enhanced or attenuated by the sensing of intracellular bacteria.
Murine RAW 264.7 cells were rendered endotoxin-tolerant by pre-exposure to heat-killed Salmonella typhimurium SL1344 ("HKS") at an MOI of 10 for 20 h. 5 days upon removal of the HKS stimulus cells were exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication. The wild-type Salmonella strain invades and replicates in endotoxin-tolerant RAW 264.7 cells, though the intracellular replication rate is significantly reduced (by roughly 40 %) compared to naive RAW 264.7 cells.
We compared mouse mRNA abundances between cells infected with wild-type ("wt") and non-infected, mock-treated controls ("Mock") or between wild-type ("wt") and SPI1/2-knockout ("SPI1/2") infected cells. As apparent from the Microarray data the wild-type Salmonella strain induces a typical inflamatory response in endotoxin-tolerant RAW 264.7 cells (as judged from the fold-induction of NFkB-inducible genes). As expected however, mRNAs corresponding to known inflammatory genes are significantly less abundant in response to the non-invasive SPI1/2 knockout strain, compared to wild-type infection. These data indicate, that endotoxin-tolerance renders macrophages largely insensitive to extracellular, but not to intracellular bacteria.

***This submission represents the mRNA component of the study

first: Leon

last: Schulte

first: Jörg

last: Vogel

first: Ana

last: Eulalio

first: Richard

last: Reinhardt


title: Drosophila_20min_courtship

status: Public on Jan 21 2011

city: College Station

first: Ginger

last: Carney

state: TX

address: 3258 TAMU

department: Biology

institute: Texas A&M University

summary: We hypothesized that social interactions, such as those involved in reproductive behaviors, would lead to immediate and assayable changes in gene expression that may have important effects on individual reproductive success and fitness through alterations in physiology or via short-term or long-term changes in nervous system function.

overall_design: Five-day-old males were exposed or mock-exposed to a virgin female fly for 20 min. Exposed males that courted, as well as mock-exposed males, were collected for RNA extraction of head tissue. Five samples for each treatment were hybridized to Affymetrix Drosophila Genome arrays for a total of 15 arrays. Data were analyzed using dChip PM, dChip PM-MM, MAS5.0, R/Bioconductor GC-RMA, and GeneSpring GC-RMA.

middle: L

last: Ellis

middle: E


title: An ENU-induced point mutation in the mouse Btaf1 gene causes post-gastrulation embryonic lethality and protein instability

city: Utrecht

first: Frits

last: Meijlink

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary: The mouse Btaf1 gene, an ortholog of yeast MOT1, encodes a highly conserved general transcription factor.  The function of this SNF-2 like ATPase has been studied mainly in yeast and human cells, which has revealed that it binds directly to TBP, forming the B-TFIID complex. This complex binds to core promoters of RNA polymerase II- transcribed genes and, of crucial importance, BTAF1-TBP interactions have been shown to affect the kinetics of TBP-promoter interactions. Here we report the isolation of a mouse line carrying a Btaf1 allele containing an ENU-induced point mutation that causes a substitution mutation in the BTAF1 ATPase domain. Embryos homozygous for this loss-of-function mutation appear to be morphologically normal until early somite stages, but die between embryonic day 9 and 10.5 displaying growth arrest and edema.  Analyses in vitro suggest that the altered protein is less stable and, independent from this, functionally impaired in releasing of TBP from chromatin, but not in binding to TBP.

overall_design: A microarray screen for genes with altered expression in the Btaf V1330M mutant as compared to WT embryos was performed with RNA from whole embryos. We compared mRNA transcripts from pools of Btaf1-/- and Btaf1+/+ embryos. Where possible, we used 3-5-somite embryos, a stage we considered to be immediately prior to the emergence of a perceptible phenotype. The experiments were done in duplo, i.e. two sets of comparisons were made:  in one case stage matching (i.e. number of somites counted) was the highest priority, in the other case the pools were designed such that a maximum number of litter mates were compared while a difference of up to five somites was allowed.  cDNAs were synthesized and Cy-3/Cy-5 labeled cRNAs were generated by using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Santa Clara, CA). The labeled cRNAs were hybridized on 4X44K Agilent Whole Mouse Genome Dual Color Microarrays (G4122F). Two dye-swap experiments were performed, resulting in four individual arrays. Microarray signal and background information were retrieved with Feature Extraction (V9.5.3, Agilent technologies). All data analyses were performed by using ArrayAssist (5.5.1, Stratagene Inc, La Jolla, CA) and Microsoft Excel (Microsoft Corporation, Redmont, WA). Genetic background: C57BL/6J were mutagenized, and crossed against FVB/NJ mice for positional  mapping. The background of the mice used in the experiment is hybrid C57BL/6J *FVB/NJ with a percentage FVB between 50 and 90%.

last: van Gurp

first: Carolien

last: Wansleeben


title: Regulation of the anti-immune response by Chlorella intake during swimming stress in mice

status: Public on Jan 19 2011

city: Kyoto

first: Toru

last: Mizoguchi

state: Kyoto

address: 369, Karasuma-dori, Gojo, Shimogyo-ku

department: Research and Development Department

institute: Sun Chlorella Corp.

summary: Chlorella has been used as a food supplement and has been reported to have health benefits. We used the forced swimming test to investigate the influence of Chlorella intake during muscle stress training in mice. Swimming time was about two-fold longer for the Chlorella intake mice than for the control mice. Microarray analysis revealed that the global expression profile of muscle from the Chlorella intake mice was similar to that for intact (nonswimming) mice rather than to that for control swimming mice. By contrast, oxidoreductase activity and the leukotriene synthesis pathway were repressed in Chlorella intake mice. According to measurement of the cytokine activities of primary cultures of spleen, Th1 cytokines, such as interferon-g・and interleukin-2, and granulocyte colony-stimulating factors, such as MIP-1a・ GMCSF, and IP-10, were decreased in control swimming mice, but these cytokines were rescued in the Chlorella intake mice. We suggest that the immune response during muscle training can be repressed by intake of Chlorella.

overall_design: The intact (nonswimming) and control (swimming) groups were fed normal food, and the Chlorella intake (swimming) group was fed food including 0.5% Chlorella powder. After 14days, the total RNA was isolated from muscle and samples was pooled for each group (n = 10 for each group).

first: Yukari

last: Arakawa

first: Masaki

last: Fujishima


title: Identification of genes that elicit disuse muscle atrophy via the transcription factors p50 and Bcl-3

status: Public on Jan 10 2011

city: Boston

first: Susan

last: Kandarian

address: 635commonwealth ave, Rm444

department: Health Sciences

institute: Boston University

summary: Skeletal muscle atrophy is a debilitating condition associated with weakness, fatigue, and reduced functional capacity. Nuclear factor-kappaB (NF-κB) transcription factors play a critical role in atrophy.  Knockout of genes encoding p50 or the NF-κB co-transactivator, Bcl-3, abolish disuse atrophy and thus they are NF-κB factors required for disuse atrophy.  We do not know however, the genes targeted by NF-κB that produce the atrophied phenotype.  Here we identify the genes required to produce disuse atrophy using gene expression profiling in wild type compared to Nfkb1 (gene encodes p50) and Bcl-3 deficient mice.  There were 185 and 240 genes upregulated in wild type mice due to unloading, that were not upregulated in Nfkb1-/- and Bcl-3-/- mice, respectively, and so these genes were considered direct or indirect targets of p50 and Bcl-3.  All of the p50 gene targets were contained in the Bcl-3 gene target list.  Most genes were involved with protein degradation, signaling, translation, transcription, and transport.  To identify direct targets of p50 and Bcl-3 we performed chromatin immunoprecipitation of selected genes previously shown to have roles in atrophy.  Trim63 (MuRF1), Fbxo32 (MAFbx), Ubc, Ctsl, Runx1, Tnfrsf12a (Tweak receptor), and Cxcl10 (IP-10) showed increased Bcl-3 binding to κB sites in unloaded muscle and thus were direct targets of Bcl-3.  p50 binding to the same sites on these genes either did not change or increased, supporting the idea of p50:Bcl-3 binding complexes.  p65 binding to κB sites showed decreased or no binding to these genes with unloading.  Fbxo9, Psma6, Psmc4, Psmg4, Foxo3, Ankrd1 (CARP), and Eif4ebp1 did not show changes in p65, p50, or Bcl-3 binding to κB sites, and so were considered indirect targets of p50 and Bcl-3.  This work represents the first study to use a global approach to identify genes required to produce the atrophied phenotype with disuse.

overall_design: 24 mice were used based on 4 mice per group, 3 mouse genotypes (wild type, Nfkb1-/-, Bcl3-/-) and 2 conditions (weight-bearing and unloaded).

first: Chia-Ling

last: Wu

middle: C

first: Robert

last: Jackman


title: Sirt1 targeting promotes Treg function

status: Public on Jan 05 2011

city: Philadelphia

first: Wayne

last: Hancock

address: 916B ARC, 3615 Civic Ctr Blvd

institute: CHOP/UPenn

summary: Sirtuin-1 (Sirt1), a class III histone/protein deacetylase is central to cellular metabolism, stress responses and aging, but its contributions to various host immune functions have been little investigated. To study the role of Sirt1 in T-cell functions, we undertook targeted deletions by mating mice with a floxed Sirt1 gene to mice expressing CD4-cre or Foxp3-cre recombinase, respectively. We found that Sirt1 deletion left conventional T-effector cell activation, proliferation and cytokine production largely unaltered. However, Sirt1 targeting promoted the expression and acetylation of Foxp3, a key transcription factor in T-regulatory (Treg) cells, and increased Treg suppressive functions in vitro and in vivo. Consistent with these data, mice with targeted deletions of Sirt1 in either CD4+ T-cells or Foxp3+ Treg cells exhibited prolonged survival of MHC-mismatched cardiac allografts. Allografts in Sirt1 targeted recipients showed long-term preservation of myocardial histology and infiltration by Foxp3+ Treg cells. Comparable results were seen in wild-type allograft recipients treated with Sirt1 inhibitors, such as EX-527 and splitomicin. Hence, Sirt1 may inhibit Treg functions and its targeting may have therapeutic value in autoimmunity and transplantation.

overall_design: RNA from three independent samples from magnetically separated CD4+CD25+ Treg of Sirt1 knock out, compared to wild type (C57BL6) control

first: Ulf

last: Beier

first: Liqing

last: Wang

first: Tricia

last: Bhatti

first: Yujie

last: Liu

first: Rongxiang

last: Han

first: Guanghui

last: Ge


title: Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and their denucleation [eyeball tissue]

status: Public on Jan 01 2011

city: LYON

first: Jiri

last: ZAVADIL

address: 150 COURS ALBERT THOMAS

institute: IARC WHO

summary:
Genome-wide approach to identify the cell-autonomous role of Brg1 in lens fiber cell terminal differentiation.
Differential gene expression was analyzed in Brg1 lens-conditional knockout and wildtype newborn mouse eyeballs, with subsequent comparison of  this data  with the dnBrg1 mouse lenses expression data.

Keywords: Differential gene expression

overall_design: Three biological replicate experiments were performed.

first: Shuying

last: He

first: Melinda

last: Pirity

first: Wei-Lin

first: Louise

last: Wolf

first: Bharesh

middle: K

last: Chauhan

first: Kveta

last: Cveklova

first: Akira

last: Nakai

first: Ernest

last: Tamm

first: Ruth

last: Ashery-Padan

last: Zavadil

first: Ales

last: Cvekl


title: Global profiling of gene expression in mouse astrocyte in response to the potential longevity determinant miR-29

status: Public on Feb 08 2011

city: Nara City

first: Hiroyuki

last: Tanabe

state: Nara

address: 3327-204 Nakamachi

department: Faculty of Agriculture Department of Advanced Bioscience

institute: Kinki University

summary: MicroRNAs (miRNAs) are an evolutionarily conserved large class of small non-coding RNAs that mediate post-transcriptional silencing of genes and influence a broad spectrum of biological processes ranging from embryonic development to organismal death. Our previous study identified the miR-29 family, three paralogous species of miR-29a/b/c, as the most predominantly expressed small RNA in aged mouse brain compared to the neonate one. Mouse brain miR-29 is highly astrocytic. Its expression is quiescent during early brain development, and then steadily increases to a plateau state around reproductive maturity. To explore the functional relevance of miR-29 expression in early life to the neural physiology of the mouse brain from the mechanistic perspective of mammalian species-specific lifespan, we here undertook a gain-of-function approach through exogenous expression of miR-29 in astrocyte from mouse fetus and surveyed the resulting alteration in both the transcriptional and translational levels. DNA microarray analyses retrieved a total of 5,589 genes showing temporal significant expression changes in the miR-29-transfected fetal astrocytes, and classified them into two gene groups: positively or negatively regulated by miR-29. Mass spectrometry (MS)-based quantification of translational products of miR-29-responsive genes identified 18 species of miR-29 target candidates. We performed functional enrichment analyses utilizing bioinformatics resources to characterize the gene sets thus identified, and found their expression trend that favor the processes for facilitating cell differentiation while supporting normal cell proliferation /survival, which is somewhat different from the functional signatures of miR-29 as observed at adult stages, implying a pleiotropic property of miR-29 depending on the developmental context. Our present results strongly suggest that miR-29 in the developing mouse brain serves as the central coordinator to shift the global gene expression toward adult phenotypes, through which ensure the programmed transition in the life course to the post-developmental/reproductive stage which has inherently been set to delimit the mouse life potential.

overall_design: DNA microarray experiment: Sample labeling, hybridization and washing were performed following the standard protocol detailed in the Agilent One-Color Microarray-Based Gene Expression Analysis ver.5.7. Briefly, a One-color Spike-Mix was diluted 1000-fold and a 5-μl aliquot of the diluted mix were added to every 0.5 μg of total RNA samples (three each of extraction replicates of miR-29-treated sample and control sample named elsewhere) prior to labeling reaction. The labeling reaction was carried out separately for the RNA samples using a Quick Amp Labeling Kit one-color, in the presence of cyanine 3-CTP. The dye-labeled target (1.6 μg as cRNA) was fragmented and hybridized on an Agilent Whole Mouse Genome 4X44K microarray at 65°C for 17 hr with a Gene Expression Hybridization Kit. The hybridized slide was washed in Gene Expression Wash Buffer 1 at room temperature for 1 min, which was followed by a wash for 1 min in Gene Expression Wash Buffer 2. The processed microarrays were scanned using an Agilent DNA Microarray Scanner. Data extraction from raw image files was done with Agilent Feature Extraction software ver.10.7.


title: Global changes in gene expression in response to miR-200 overexpression in 4TO7 cells

status: Public on Dec 23 2010

city: Princeton

first: Yibin

last: Kang

state: NJ

address: LTL 254, Washington Road

department: Molecular Biology

institute: Princeton University

summary: Transcriptional profiling of 4TO7 cells stably overexpressing miR-200s or CDH1. We observe a dramatic shift in the gene signatures when miR-200s (cluster 2; miR-200c/141, or both clusters) are overexpressed relative to controls. However, cluster 1 overexpression (miRs-200b/a/429) alone or CDH1 overexpression does not induce global changes in gene expression to levels observed for cluster 2 (miR-200c/141) or both clusters together.

overall_design: One cell line (4TO7) cells: four different overexpression experiments, Cluster 1, Cluster 2, Clusters 1+2, CDH1, and respective controls, Puro vec, Hygro vec, Puro+Hygro vec and Puro vec. Each experiment has two biological replicates. Total, 16 samples.

first: Manav

last: Korpal