title: mRNA composition of IRP1 mRNPs in mouse tissues

summary: Affymetrix microarrays were used to determine the mRNA composition of mRNPs obtained by immunoprecipitation with IRP1 (iron regulatory protein 1).

overall_design:
Reference Sanchez at al., 2007 Nat. Protoc. Affymetrix microarrays were used to determine the mRNA composition of mRNPs obtained by immunoprecipitation with IRPs (iron regulatory proteins). In brief, total RNA was extracted from the reported tissue of four to six C57BL6/J mice using Trizol reagent. mRNA from duodenum, liver and spleen were extracted from mice fed with an iron poor diet (<10mg/kg, Altromin Spezialfutter GmbH & Co, Lage, Germany) for 21 days starting from weaning age. mRNAs from bone marrow and brian were extracted from mice (8-10 weeks old) fed with an iron-normal diet. For each tissue, the RNA samples were pooled and the immunoprecipitations were carried out combining 50 µg of total RNA with purified recombinant IRP1 produced in E.coli and a rabbit polyclonal anti-IRP1 antibody. A control reaction in which the recombinant IRP1 was omitted was performed in parallel (referred with the word “control” in the title).
All the microarray procedures were conducted at  the EMBL Genomics Core Facility using standard Affymetrix protocols.  In brief, approximately 120ng of immunoprecipitated RNA was used as input to a two-step amplification procedure to generate biotin-labeled RNA fragments for hybridization to the Affymetrix GeneChip Mouse Genome 430 2.0 array (Eukaryotic Sample and Array Processing manual 701024 Rev.3). Intensity values for the hybridizations were obtained either using RMA, calculations done in bioconductor  (www.bioconductor.org) or MAS5, calculated using Affymetrix GCOS package. MAS5 calculated intensities were further quantile normalized using bioconductor.


title: T cell factor 1 is a gatekeeper for T-cell specification in response to Notch signaling

status: Public on Dec 06 2011

city: Cambridge

first: Jiangwen

last: Zhang

state: MA

address: 7 Divinity Ave

department: FAS Center for Systems Biology

country: USA

institute: Harvard University

summary:
Although transcriptional programs associated with T-cell specification and commitment have been described, the functional hierarchy and the roles of key regulators in structuring/ orchestrating these programs remain unclear. Activation of Notch signaling in uncommitted precursors by the thymic stroma initiates the T-cell differentiation program. One regulator first induced in these precursors is the DNA binding protein Tcf-1, a T-cell specific mediator of Wnt signaling. Yet the specific contribution of Tcf-1 to early T-cell development and the signals inducing it in these cells remain unclear. Here we assign functional significance to Tcf-1 as a gatekeeper of T-cell fate. We show that Tcf-1 is directly activated by Notch signals. Tcf-1 is required at the earliest phase of Tcell determination for progression beyond the early thymic progenitor (ETP) stage. The global expression profile of Tcf-1 deficient progenitors indicates that basic processes of DNA metabolism are downregulated in its absence and the blocked T-cell progenitors become abortive and die by apoptosis. Our data thus add an important functional relationship to the roadmap of T-cell development.
We used microarrays to detail the global programme of gene expression of mouse ETP thymocyte after Ikaros inactivation with dominant negative of Ik at different stage.

overall_design: 6 samples (mouse ETP thymocytes from wt, and Tcf1 inactivation) are analyzed Mouse Microarray Expression platforms, Affymetrix Mouse 430 2.0,

first: Kristine

last: Germar

first: Marei

last: Dose

first: Fotini

last: Gounari


title: Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice [Expression array].

status: Public on Dec 14 2011

city: Basel

first: Jonathan

last: Moggs

address: Fabrikstrasse 2

country: Switzerland

institute: Novartis

summary: Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.

overall_design: 29–32 days old male B6C3F1/Crl (C57BL/6 ♂ x C3H/He ♀) mice were obtained from Charles River Laboratories (Germany). Animals were allowed to acclimatise for 5 days prior to being randomly divided into two treatment groups (n = 10) and phenobarbital (Sigma 04710, 0.05% (w/v) in drinking water) was administered to one group through ad libitum access to drinking water for 28 days. Mice were checked daily for activity and behavior and sacrificed on the last day of dosing (day 28). Blood was withdrawn for PK analysis and target (liver) and non-target (kidney) tissues removed, split into several sections, frozen in liquid nitrogen and stored at −80°C for subsequent analyses. Total RNA from liver and kidney was purified and processed for Affymetrix gene expression profiling while genomic DNA was prepared for promoter array based methylome analysis using the Methylated DNA immunoprecipitation (MeDIP) procedure. Remaining tissue material was used for chromatin immunoprecipitation (ChIP) to analyze histone modifications at individual promoters. Plasma samples were also collected to evaluate phenobarbital exposure in individual animals by LC-MS.

first: Harri

last: Lempiäinen

first: Arne

last: Müller

first: Sarah

last: Brasa

first: Soon-Siong

last: Teo

first: Tim-Christoph

last: Roloff

first: Laurent

last: Morawiec

first: Natasa

last: Zamurovic

first: Axel

last: Vicart

first: Enrico

last: Funhoff

first: Philippe

last: Couttet

first: Dirk

last: Schübeler

first: Olivier

last: Grenet

first: Jennifer

last: Marlowe

first: Rémi

last: Terranova


title: Identification of novel androgen-regulated pathways and mRNA isoforms through genome-wide exon-specific profiling of the LNCaP transcriptome

status: Public on Dec 27 2011

city: Newcastle-upon-Tyne

first: Prabhakar

last: Rajan

address: Central Parkway

department: Institute of Human Genetics

country: United Kingdom

institute: Newcastle University

summary: Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity.  Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome.  While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes.  Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer.  We also identified 1279 putative androgen-regulated alternative events, of which 325 (~25%) mapped to known alternative splicing events or alternative first/last exons.  We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators.  Of seven positively-validating events (~23%), five events involved transcripts derived from known AR gene targets.  In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition.  We confirmed that expression of the alternative TSC2 mRNA isoform was directly regulated by androgens.  Furthermore, by chromatin immunoprecipitation, we observed recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts.  Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa.

overall_design: Total 8 samples were analysed. 4 control (LNCaP cells grown in RPMI-1640 media with charcoal-stripped 10% FBS devoid of steroids) and 4 treatment (LNCaP cells grown in media devoid of steroid, and subsequently treated with 10nM R1881 synthetic androgen analogue for 24 hours).

first: Caroline

last: Dalgliesh

first: Phillippa

last: Carling

first: Thomas

last: Buist

first: Chaolin

last: Zhang

first: Sushma

middle: N

last: Grellscheid

first: Kelly

last: Armstrong

first: Jacqueline

last: Stockley

first: Cedric

last: Simillion

first: Luke

last: Gaughan

first: Gabriela

last: Kalna

first: Michael

middle: Q

first: Craig

last: Robson

first: Hing

middle: Y

last: Leung

first: David


title: Functional and epigenetic studies reveal multistep differentiation and plasticity of in vitro and in vivo follicular T helper cells.

city: BETHESDA

first: abdel

middle: G

last: Elkahloun

address: 50, SOUTH DRIVE

institute: NHGRI-NIH

summary:
Follicular T helper cells (Tfh) are critical for providing help to B cells for germinal center (GC) formation. Mutations affecting SAP prevent GC formation due to defective T:B cell interactions, yet effects on Tfh cell differentiation remain unclear. We describe the in vitro differentiation of functionally competent “Tfh-like” cells that expressed IL-21, Tfh markers, and Bcl6, and rescued GC formation in SAP-deficient hosts substantially better than other T helper (Th) cells. SAP-deficient Tfh-like cells appeared virtually indistinguishable from wildtype, yet failed to support GCs in vivo. Interestingly, both Tfh-like and in vivo-derived Tfh cells could produce effector cytokines in response to polarizing conditions. Moreover, other Th cell populations could be reprogrammed to obtain Tfh characteristics. ChIP-Seq analyses revealed positive epigenetic markings on Tbx21, Gata3 and Rorc in Tfh-like and ex vivo Tfh cells, and Bcl6 in other Th cells, supporting the concept of plasticity between Tfh and other Th populations.
We describe the in vitro differentiation of functionally competent IL-21-producing cells with Tfh-like properties. Importantly, transfer of low numbers of these cells induced GC formation in SAP-deficient hosts more effectively than other in vitro differentiated Th cells, suggesting they represent bona fide Tfh cell precursors. We have chosen the name “Tfh-like” cells for these in vitro differentiated IL-21 producing cells as they exhibit Tfh characteristics, but do not reside within B cell follicles. SAP-deficient Tfh-like cells were virtually indistinguishable from WT, yet nonetheless, failed to effectively contribute to Tfh cells and rescue GC formation in vivo. Evaluation of cytokine production as well as epigenetic chromatin modifications of genes encoding Th cell-specific transcription factors from either in vitro-generated Tfh-like cells or Tfh cells isolated directly ex vivo provided evidence for plasticity between Tfh-like and other Th cell populations. Our results provide insight into the requirements for differentiation and plasticity of Tfh cells, which are critical for the generation of effective long-term humoral immunity.

overall_design: RNA was prepared from subconfluent asynchronously proliferating cells using TRIZOL and purified by RNeasy MinElute Cleanup kit (Qiagen).  Hybridization to Affymetrix GeneChip Mouse Genome 430 2.0 arrays was used to generate gene expression profiles of WT Tfh-like and SAP-deficient Tfh-like cells.

first: Kristina

middle: T

last: Lu

first: Abdel

first: Pamela

middle: L

last: Schwartzberg

first: Weiwei

last: Wu


title: Genome wide analysis of acral melanoma (Illumina)

status: Public on Dec 13 2011

city: London

first: Simon

last: Furney

address: 237 Fulham Road

institute: Institute of Cancer Research

summary:
Assessment of mutation on expression levels
Transcriptomic profile of a matched  primary and metastatic acral melanoma

overall_design: One Primary and one metastatic acral melanoma transcript expression were assayed (no matched normal)

first: S

middle: J

last: Turajlic


title: Transcriptional profiling of MMTV-tTA/TOP-ICN1 tumor derived cell lines comparing untreated (ICN1-On) cells to 24-hour doxcycline treated (ICN1-Off) samples

status: Public on Dec 05 2011

city: Worcester

first: Matthew

last: Simmons

address: 364 Plantation St

department: Cancer Biology

institute: UMASS Medical School

summary: NOTCH activation has been recently implicated in human basal-like breast cancers associated with a poor prognosis. To address the role of Notch1 in mammary transformation and mammary tumor initiating cell activity, we developed a doxycycline-regulated model of Notch1-mediated mammary transformation. These mice develop mammary adenocarcinomas that express cytokeratin (CK) 8/18 and contain rare cells that also express keratin 14. In vivo limiting dilution analyses reveal that these mammary tumors exhibit functional heterogeneity and harbor a rare (1/2978) mammary tumor initiating cell population. Using this dox-regulated Notch1 mammary tumor model, we demonstrate that Notch1 inhibition results in mammary tumor regression in vivo and prevents disease recurrence in 4 of 6 tumors tested. Consistent with the in vivo data, Notch1 inhibition reduces mammary tumorsphere forming activity in vitro. Using doxycycline-responsive tumor derived cell lines, we also identify the embryonic stem cell transcription factor Nanog as a novel Notch1-regulated gene in mammospheres. These data indicate that Notch1 contributes to mammary tumor initiating activity and raises the possibility that NOTCH therapeutics may have efficacy in human basal-like breast cancers associated with NOTCH activation.

overall_design: Primary mammary tumors were isolated from two different MMTV-tTA/TOP-ICN1 transgenic mice, minced, enzymatically digested and converted to culture.  To identify changes in gene expression in response to ICN1 suppression, tumor-derived cell lines 8534 and 8542 were left untreated (8534-Untreated; 8542-Untreated) or treated with 2ug/ml doxycycline for 24 hours (8534-Dox; 8542-Dox).  Cells were collected by scraping and total RNA was isolated, followed by real-time PCR validation of NOTCH1 target gene modulation.  RNA samples were further hybridized to Affymetrix mouse genome 430A2.0 arrays.

first: Michelle

middle: A

last: Kelliher


title: Vascular endothelial growth factor (VEGF) isoform regulation of early forebrain development

status: Public on Dec 01 2011

city: Grand Forks

first: Diane

middle: Catherine

last: Darland

state: ND

address: 10 Cornell Street/Campus Drive

department: Department of Biology

institute: University of North Dakota

summary: This work was designed to determine the role of the vascular endothelial growth factor A (VEGF) isoforms during early neuroepithelial development in the mammalian central nervous system (CNS), specifically in the forebrain.  An emerging model of interdependence between neural and vascular systems includes VEGF, with its dual roles as a potent angiogenesis factor and neural regulator.  Although a number of studies have implicated VEGF in CNS development, little is known about the role that the different VEGF isoforms play in early neurogenesis. We used a mouse model of disrupted VEGF isoform expression that eliminates the predominant brain isoform, VEGF164, and expresses only the diffusible form, VEGF120.  We tested the hypothesis that VEGF164 plays a key role in controlling neural precursor populations in developing cortex. We used microarray analysis to compare gene expression differences between wild type and VEGF120 mice at E9.5, the primitive stem cell stage of the neuroepithelium. We quantified changes in PHH3-positive nuclei, neural stem cell markers (Pax6 and nestin) and the Tbr2-positive intermediate progenitors at E11.5 when the neural precursor population is expanding rapidly. Absence of VEGF164 (and VEGF188) leads to reduced proliferation without an apparent effect on the number of Tbr2-positive cells.  There is a corresponding reduction in the number of mitotic spindles that are oriented parallel to the ventricular surface relative to those with a vertical or oblique angle. These results support a role for the VEGF isoforms in supporting the neural precursor population of the early neuroepithelium.

overall_design: Four samples each of E9.5 wildtype or VEGF120 mouse forebrain were analyzed with the Mouse 430 2.0 Affymetrix GeneChip

middle: C

first: Jacob

last: Cain

last: Berosik

first: Magali

last: Saint-Geniez

first: Patrick

middle: W

last: Odens

first: Geoffrey

last: Schaubhut

first: Sarah

last: Frisch

first: Anat

last: Stemmer-Rachamimov

first: Tristan

first: Patricia

last: D'Amore


title: Expression profiling of the early postnatal stage of Polycystic Kidney Disease in the B6C3Fe a/a-bpck mice

city: Tianjin

first: E

last: Du

address: ping jiang road 23#, Hexi district

country: China

institute: Tianjin institude of Urology

summary:
To understand the molecular and cellular mechanisms of pathogenesis of autosomal recessive polycystic kidney disease , we performed a microarray gene expression profiling in early stage kidneys  of B6C3Fe a/a-bpck mutant and wild-type mice at postnatal day 3.
Genes with over 1.5-fold expression changes in mutant kidneys compared with age matched wild-type tissues were selected for analysis. This study represents the first widespread profiling of B6C3Fe a/a-bpck mutant mouse kidney and provides a valuable platform for better understanding the molecular mechanisms of polycystic kidney disease in human.

overall_design: Mutational analysis was performed in accordance with the protocol of Jackson Laboratory, total RNA was extracted from kidneys of 3 days of postnatal age using a monophasic solution of phenol/guanidine isothiocyanate and TRIzol reagent (Invitrogen) according to their manual, and the samples were incubated with RNase-free DNase I (Ambion). The quality and concentration of each sample were confirmed by spectrophotometry.  Affymetrix 430 2.0 arrays were used according to standard Affymetrix procedures. Data analysis was performed with dChip software (Dec.2010 version). These measurements were confirmed by Real-Time PCR.

first: Ruifa

last: Han

first: Hong

last: Li

first: Shunying

last: Jin

first: Kang

last: Zheng

first: Xiaofeng

last: Zhao

first: Yanchun

last: Qu

first: Xuemei

last: Hu

first: Qiang

last: Zhu

first: Binghua

last: Xie

first: Mengsheng

last: Qiu


title: An Animal Model of Myc-driven medulloblastoma

city: SAN DIEGO

first: Alexey

middle: M

last: Eroshkin

state: California

address: Alexey Eroshkin, 3803 Ruette San Raphael

department: Bioinformatics Core

institute: SBPMDI

summary:
Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB, and identify a novel model that can be used to test therapies for this devastating disease.
To gain insight into the pathways that control growth of MYC-driven MB, we compared gene expression profiles of murine Myc/DNp53 (MP) tumor cells to those of freshly isolated cerebellar stem cells (Prom1+Lin- cells) and of tumors from Ptch1 mutant mice (a model for Sonic Hedgehog-associated MB). RNA was isolated from stem cells and tumor cells using the RNAqueous kit (Ambion). RNA was labeled and hybridized to Affymetrix Mouse Genome 430 2.0 arrays.

overall_design: 19 mouse cell samples (stem cells and tumor cells) were analyzed. There are four groups of samples, three with five biological replicates and the last with four (one outlier was removed).  To gain insight into the mechanisms of transformation into tumors, we compared the gene expression profiles of MP tumor cells derived from stem cells (Myc/DNp53-infected Prom1+Lin- cells, designated MP-pl) or progenitors (Myc/DNp53-infected Prom1+ cells, designated MP-p) to gene expression profiles of uninfected stem cells (designated NSC) and profiles from a distinct model of medulloblastoma, the patched mutant mouse (designated ptch1).

first: Robert

last: Wechsler-Reya

first: Yanxin

last: Pei


title: Expression data from the liver of wild-type and Cnot3+/- mice: Fed vs Fasted

status: Public on Dec 09 2011

city: San Antonio

first: Masahiro

last: Morita

state: Texas

address: 4939 Charles Katz Drive

department: Department of Molecular Medicine

institute: University of Texas Health Science Center at San Antonio

summary:
Decay of mRNAs initiates with shortening of the poly(A) tail. Although the CCR4-NOT complex participates in deadenylation, how it becomes activates remain obscure. We show that complete deficiency in CNOT3, subunit 3 of this complex, is lethal in mice, but that heterozygotes survive as lean mice with hepatic and adipose tissues containing reduced lipid levels. Cnot3+/- mice have enhanced metabolic rates and remain lean on high-fat diets. We further provide evidence suggesting that CNOT3, by changing its level in response to feeding conditions, affects the activity of the CCR4-NOT deadenylase against poly(A) tails of specific mRNAs coding for proteins involved in metabolism of carbohydrates and fats.
Because the levels of CNOT3 protein were decreased under fasting conditions and increased upon refeeding and because CNOT3 could be a positive regulator of the CCR4-NOT deadenylase, we hypothesized that the levels of CCR4-NOT target mRNAs would be lower in fed mice than in fasted mice. We compared the gene expression profiles of fed and fasted wild-type mice. Microarray analysis revealed that approximately 1,200 mRNA transcripts were down-regulated in the livers of fed mice. Of these mRNAs, 68 corresponded to the genes up-regulated in the livers of Cnot3+/- mice and fasted wild-type mice. A large number of the 68 identified genes encoded proteins involved in metabolism, especially lipid metabolism and growth.

overall_design: The livers were isolated from 8-week-old fed wild-type, fasted wild-type and fasted Cnot3+/- mice (n = 2 for each genotype).

first: Takeshi

last: Nagashima


title: Lineage relationship in human dendritic cells

city: Melbourne

first: Christopher

last: Bye

state: Victoria

address: University of Melbourne

country: Australia

institute: The Florey Institute of Neuroscience and Mental Health

summary: The lineage relationships and fate of human blood and tissue dendritic cells (DC) has significance for a number of diseases including HIV where both blood and tissue DC may be infected. We used gene expression profiling of monocyte and DC sub-populations sorted directly from blood and skin and compared this to monocyte derived DC (MDDC) and MUTZ3 Langerhans cells (LCs) to define the lineage relationships. Hierarchical clustering analysis showed that plasmacytoid DCs formed the most discrete cluster. The ex vivo derived myeloid cells formed two separate clusters of cells derived from blood, and skin. Separate and specific DC populations could be determined within the sub-clusters. During overnight culture CD14+ dermal DCs (DDC) converted to CD1a+ expressing cells in situ consistent with origin of the CD1a+ DDC from a local precursor rather than from circulating blood DC or monocyte precursors. The in vitro derived MDDC and MUTZ3 populations grouped within the skin DC cluster and MDDCs clustered most closely to CD14+ DDC consistent with the proposed similarity between these two cell types. We identified differential expression of novel genes in particular DC subsets including genes related to DC surface receptors (including C-type lectin receptors, toll-like receptors and galectins).

overall_design: Total RNA was extracted and hybridised to 24 bead arrays. Dendritic cells and monocytes from human blood and skin using magnetic bead and flow cytometry based cell sorting both before and after culture for 24 hours

first: Andrew

last: Harman

first: Chris

middle: R

first: Kerrie

last: Sandgren

first: Sarah

middle: K

last: Mercier

first: Najla

last: Nasr

first: Sharon

last: Lewin

first: Anthony

last: Cunningham

first: Paul

middle: U

last: Cameron


title: Resection of Non-Small Cell Lung Cancers Reverses Tumor-Induced Gene Expression Changes in the Peripheral Immune System

city: Philadelphia

first: Louise

last: Showe

state: PA

address: 3601 Spruce St

institute: The Wistar Institute

summary: We have examined the changes that occur in microRNA (miRNA) expression levels in peripheral blood mononuclear cells (PBMC) collected from  paired pre- vs. post-tumor removal samples in order to characterize the way lung tumor removal affects gene expression in peripheral immune cells.  5 miRNAs were expressed at significantly higher levels before vs. after tumor removal. Significant number of genes that were identified computationally as targets of the miRNAs and negatively correlated with the miRNA expression were transcription factors.

overall_design: miRNA expression changes were compared between 11 pre and post surgery NSCLC lung cancer samples.

middle: V

last: Kossenkov

first: Anil

last: Vachani

first: Celia

last: Chang

first: Calen

last: Nichols

first: Shere

last: Billouin

first: Wenhwai

last: Horng

first: William

last: Rom

first: Steven

last: Albelda

first: Michael


title: Differential expression of genes after transfection of STAT6 specific siRNA in NCI-H460 cells

city: Delhi

first: Neeru

last: Saini

address: Mall Road

department: FGU

country: India

institute: IGIB

summary: The experiment was designed to identify the genes which get altered after the transfection of siRNA targeting STAT6 in NCI-H460 cells. The NCI-H460 cells plated in 12-well plate were transfected with 60nM of STAT6 specific siRNA.

overall_design: Biological duplicates of 2 samples were used viz. control NCI-H460 cells (Contol), NCI-H460 cells transfected with siRNA (siRNA)

first: Richa

last: Dubey

first: Ravindresh

last: Chhabra


title: Early responding dendritic cells direct local natural killer response to control HSV-1 infection within the cornea

status: Public on Nov 30 2011

city: Pittsburgh

first: Stephen

middle: Alan Kenneth

last: Harvey

address: 203 Lothrop St

department: Ophthalmology

institute: University of Pittsburgh School of Medicine

summary:
Dendritic cells (DCs) regulate both innate and adaptive immune responses.
The role of CD11c-plus DCs in the corneal response to to herpes simplex virus-1 (HSV-1) infection was investigated by depleting them prior to infection.

overall_design:
Bone marrow cells from CD11c-DTR (C.FVB-Tg(Itgax-DTR/EGFP)57Lan/J) mice were transferred intravenously to irradiated BALB/cJ host mice.
After 6 weeks, the bone marrow chimeric host mice were injected with diphtheria toxin (DT) to deplete CD11c-positive cells.
Two days after injection, the mice were subjected to a standard corneal infection protocol using HSV-1.

first: Gregory

last: Frank

first: Kristine-Ann

last: Buela

first: Dawn

last: Maker

first: Robert

last: Hendricks


title: Gene Expression Changes Associated with the Progression of Intraductal Papillary Mucinous Neoplasms

city: Royal Oak

first: Bryan

last: Thibodeau

state: MI

address: 3811 West 13 Mile Road

department: BioBank

institute: Beaumont Research Institute

summary:
Purpose: The diagnosis of high grade intraductal papillary mucinous neoplasm (IPMN) is difficult to distinguish from low grade IPMN. The aim of this study was to identify potential markers for the discrimination of high grade and invasive IPMN from low and moderate grade IPMN.
Experimental Design: Laser capture microdissection was used to isolate distinct foci of low grade, moderate grade, high grade, and invasive IPMN from paraffin embedded archival tissue from 14 patients who underwent resection for IPMN. Most samples included multiple grades in the same specimen. Affymetrix Human Exon microarrays were used for gene expression analysis to compare low and moderate grade (LMD) IPMN to high grade and invasive (HgInv) IPMN.
Results: When comparing samples of LMD dysplasia (n=15) to those demonstrating HgInv (n=13), 62 genes were identified as showing significant changes in expression (p≤0.05 and a 2-fold cutoff), with up-regulation of 41 in HgInv IPMN and down-regulation in 21. Changes in gene expression are associated with biological processes related to malignant behavior including cell motion, cell proliferation, response to hypoxia, and epithelial to mesenchymal transition. Hierarchical clustering analysis demonstrated the ability of these 62 differentially expressed genes to distinguish between LMD and HgInv samples. In addition, altered signaling in several TGFβ-related pathways was exhibited in the progression of IPMN to malignancy.
Conclusions: This study identifies a set genes associated with the progression of IPMN to malignancy. These genes are potential markers that could be used to identify IPMN requiring surgical resection.

overall_design: Using laser capture microdissection, distinct foci of low (n=10), moderate (n=5), and high grade dysplasia (n=6) as well as invasive carcinoma (n=7) were isolated from paraffin embedded archival tissue of 14 patients who underwent resection for IPMN. Gene expression data from Affymetrix Human Exon 1.0ST microarrays was utilized to compare low and moderate grade IPMN to high and invasive IPMN.

middle: P

last: Jury

first: Laura

middle: E

last: Fortier

first: Timothy

last: Geddes

first: Samreen

last: Ahmed

first: Barbara

last: Pruetz

first: Maryam

last: Farinola

first: George

middle: D

last: Wilson


title: Expression data from p27delta51 MEFs cells in quiescence

city: Barcelona

first: oriol

last: bachs

email: obachs@ub.edu, raffaellapippa@ub.edu

address: Casanova 143

department: Cell Biology, Immunology and Neurosciences

country: Spain

institute: University of Barcelona

summary: Low levels of the cell cycle regulator p27Kip1 are associated with a worse outcome in many tumor types. We report here a new regulatory role of p27Kip1 as a transcriptional regulator. In association with transcriptional repressors such as p130, E2F4 and HDACs, p27 binds to promoters of multiple genes leading to their repression. The p27Kip1-target genes participate in RNA processing, translation, respiration and cell cycle. Remarkably, p27Kip1-target genes are over-expressed in different human tumors in tight association with a poor clinical prognosis. We also observed a clear correlation between low levels of p27Kip1 and over-expression of p27Kip1-target genes in tumors. Overall, our findings indicate new tumor suppressor roles of p27Kip1 as a transcriptional regulator of genes relevant for oncogenesis.

overall_design: Samples from MEFs p27delta51 and p27WT cells used as control were analyzed

first: Raffaella

last: Pippa

first: Lluis

last: Espinosa

first: Gunes

last: Gundem

first: Ramón

last: García-Escudero

first: Ana

last: Domínguez

first: Cristina

last: Sainz

first: Arnaud

last: Besson

first: Maria

last: Pujol

first: Núria

last: López-Bigas

first: Jesus

last: Paramio

first: Oriol

last: Bachs


title: Differential effects of inhibition of bone morphogenic protein (BMP) signalling on T-cell activation and differentiation

first: Masahiro

last: Ono

address: 30 Guilford Street

department: Immunobiology Unit

institute: Institute of Child Health

summary: Dorsomorphin is a small molecule inhibitor of type I bone morphogenic protein receptors (BMPRs). We have found that dorsomorphin affects a wide range of T cell function. In order to obtain the bigger picture of the effects of DM in T cell activation. transcriptomic analysis was performed using mouse primary CD25-CD4+ T cells with either DM (4 μM) or vehicle in the presence or absence of stimulation by anti-CD3 and -CD28 antibodies.

overall_design: Mouse CD4+ T cells were prepared and cultured with or without stimulation by plate bound anti-CD3 mAb and soluble anti-CD28 mAb for 1 hour. Subsequently, 4 μM DM or DMSO was added and cells were incubated for 6 hours. Total RNA was isolated using Qiagen RNeasy mini kits.

first: Yumiko

last: Yoshioka


title: Discovery of Shear- and Side-specific mRNAs and miRNAs in Human Aortic Valvular Endothelial Cells

status: Public on Nov 29 2011

city: Atlanta

first: Hanjoong

last: Jo

state: GA

department: Biomedical Engineering

institute: Emory University

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

summary: Identify shear and side-specific miRNAs in Human Aortic Valvular Endothelial Cells using the following conditions: 1) fHAVEC exposed to OS (FO), 2) vHAVEC exposed to OS (VO), 3) fHAVEC exposed to LS (FL), and 4) vHAVEC exposed to LS (VL).

overall_design:
24 samples; n=6 for the following 4 groups: FO, VO, FL, and VL.
48 samples total (24 miRNA, 24mRNA)

first: Robert

last: Nerem

first: Casey

last: Holliday

first: Randall

middle: F

last: Ankeny


title: Gene expression profiling of Tgfbr2 mutant mouse models of cleft palate

city: Los Angeles

first: Joseph

middle: Gerard

last: Hacia

state: CA

address: 2250 Alcazar Street, IGM 261

department: Biochemistry and Molecular Biology

institute: University of Southern California

summary: The overall goal of this project is to investigate the role of TGF-beta signaling in palate development in order to discover candidate therapeutics for preventing and treating congenital birth defects. Here, we conducted gene expression profiling of embryonic palatal tissue from wild type mice as well as those with a neural crest specific conditional inactivation of the Tgfbr2 gene. The latter mice provide a model of cleft palate formation.

overall_design: To investigate the mechanism of cleft palate resulting from mutations in TGFBR2, we analyzed neural crest specific conditional inactivation of Tgfbr2 in mice (Tgfbr2fl/fl;Wnt1-Cre).  We performed microarray analyses using the palatal tissue of Tgfbr2fl/fl;Wnt1-Cre mice at embryonic day E13.5 (prior to palatal fusion, n=6 per genotype) and E14.5 (during palatal fusion, n=5 per genotype) to examine the genes regulated by Tgf-beta during palate formation.

first: Jun-ichi

last: Iwata

middle: G

first: Yang

last: Chai


title: The NOD/SCID Xenograft Model Provides Clinically-Relevant Insights into Glucocorticoid-Induced Gene Expression in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia

status: Public on Nov 28 2011

city: Randwick

first: Vivek

last: Bhadri

state: NSW

address: Lowy Cancer Research Centre

department: Leukaemia Biology Program

institute: Children's Cancer Institute Australia for Medical Research

summary: Introduction. Glucocorticoids are critical drugs used to treat acute lymphoblastic leukemia, and response to glucocorticoids is highly predictive of outcome. Here we report a study evaluating the NOD/SCID xenograft mouse model to investigate glucocorticoid-induced gene expression. Methods. NOD/SCID mice were inoculated with ALL-3, a glucocorticoid-sensitive xenograft, and when highly engrafted were randomised to either dexamethasone 15mg/kg or vehicle control IP. Cells were harvested at 0, 8, 24 or 48 hours thereafter, RNA was extracted and hybridised onto Illumina WG-6_V3 chips. Results. The 8 hour dexamethasone-treated timepoint had the highest number of significantly differentially expressed genes with minimal changes seen across the time-matched controls. Replicate analysis revealed that using data from 3 replicates instead of 4 resulted in excellent recovery scores of >0.9 at timepoints with high signal. When assessed at the level of pathways, gene expression changes in the 8 hour xenograft samples were similar to patients treated with glucocorticoids. Conclusions. The NOD/SCID xenograft mouse model provides a reproducible experimental model system in which to investigate clinically-relevant mechanisms of in vivo glucocorticoid-induced gene regulation in ALL; the 8 hour timepoint provides the highest number of significantly differentially expressed genes; time-matched controls are redundant and excellent recovery scores can be obtained with 3 replicates.

overall_design: At 0, 8, 24 or 48 hours, NOD/SCID xenograft mice were treated with vehicle control, or dexamethasone. We used 4 biological replicates per time point (3 at the 48hour time point), each of which went onto an Illumina HumanWG-6_V3_0_R1_11282955_A microarray. Samples from each of the 7 groups were divided across a total of 5 microarray slides

first: Mark

middle: J

last: Cowley

first: Warren

last: Kaplan

first: Toby

middle: N

last: Trahir

first: Richard

middle: B

last: Lock


title: Expression data from WT, HEB-KO and E2A-KO LY6D- CLP cells

status: Public on Nov 22 2011

city: La Jolla

first: Eva

last: Welinder

address: 9500 Gilman Drive

institute: UCSD

summary: The E-protein transcription factors E2A and HEB play important roles at several stages of hematopoiesis. However, the exact mechanism for theire action and the main targets in the LY6D negative common lymphoid progentior (CLP) compartment remains unknown. By adressing this question, we will gain important infromation regarding the early events leading to B-cell specification.

overall_design: FACS sorted LY6D negative common lymphoid progenitors from WT, HEB-KO and E2A-KO mice were subjected to RNA extraction and hybridization on Affymetrix microarrays. At least two independent sorts were performed per genotype. During each sort, cells were pooled from several bone marrows.

last: Mansson

first: Elinore

last: Mercer

first: David

last: Bryder

first: Mikael

last: Sigvardsson

first: Cornelis

last: Murre


title: Expression data from treatment-induced senescence in mouse Emu-myc B-cell lymphoma model

status: Public on Nov 21 2011

city: Berlin

first: Maja

last: Milanovic

address: Augustenburgerplatz 1

department: Hematology and Oncology

country: Germany

institute: Charite University Medicine, Berlin

summary:
Treatment induced senescence (TIS) is a terminal cell cycle arrest program, increasingly recognized as a tumor suppressor mechanism complementing apoptosis in response to standard chemotherapy regimens. In particular cells with blocked apoptotic pathways rely on senescence as the only remaining failsafe mechanism to keep the neoplastic growth in check. However, little is known about biological properties, long-term fate of senescent tumor cells and their impact on the microenvironment.
We used global gene expression profiling by microarrays to gain insight in the molecular programme underlying the treatment-induced senescence in Emu-myc transgenic B-cell lymphomas (apoptosis protected by Bcl2 overexpression), which robustly enter senescence in response to DNA-damaging anticancer agents such as Adriamycin (ADR).

overall_design: Primary lymphoma cells isolated from lymph nodes of Emu-Myc transgenic mice were used. In this model the the c-Myc oncogene is constitutively expressed in the cells of B-cell lineage, leading to spontaneous development of aggressive B-cell lymphomas, resembling Burkitt lymphoma in humans. In order to bring up the senescence as the main failsafe mechanism, primary lymphoma cells are protected from apoptosis by retroviral over-expression of a strong antiapoptotic protein Bcl2. These cells (Myc;Bcl2) massively undergo senescence upon DNA-damaging treatment. Adriamycin (ADR) is a cytostatic drug, used as a standard part of several lymphoma treatment regimens. In this study, transcriptional profiles of matched pairs of untreated vs. 5 days ADR treated Myc;Bcl2 lymphomas were analysed.

first: Clemens

middle: A

last: Schmitt

first: Michael

last: Hummel

first: Dido

last: Lenze

first: Jan

last: Dörr

first: Yong

last: Yu


title: Colorectal cancers with microsatellite instability display unique miRNA profiles

first: Juanjo

last: Lozano

address: C/ Rosselló 153

department: Plataforma de Bioinformatica

institute: CIBEREHD

summary: Analysis of microRNA expression of tumoral and non-tumoral colonic tissues. The aim of this study was to analyze the global miRNA signatures in various groups of well-characterized CRCs based on the presence of microsatellite instability (MSI).

overall_design: Total RNA from formalin-fixed paraffin-embedded tissue blocks from 4 different groups (normal colonic mucosa, Lynch syndrome tumors, sporadic MSI tumors and MSS tumors) was isolated using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion) according to manufacturer instructions. MiRNA expression profiles were analyzed using miRNA microarray platform.

first: F

last: Balaguer

first: L

last: Moreira

first: J

first: A

last: Link

first: G

last: Ramirez

first: Y

last: Shen

first: M

last: Cuatrecasas

last: Arnold

first: S

last: Meltzer

last: Syngal

first: E

last: Stoffel

first: R

last: Jover

first: X

last: Llor

last: Castells

first: C

last: Boland

last: Gironella

last: Goel


title: A Systematic Substrate Screen Links Cyclin D-Dependent Kinases to Senescence Suppression in Cancer Cells through FOXM1

status: Public on Nov 18 2011

city: Boston

first: Lars

last: Anders

address: 44 Binney Street

institute: Dana-Farber Cancer Institute

summary:
Gene expression (mRNA profiling) of U2OS osteosarcoma cells treated with 1 mM of the CDK4/6-specific inhibitor, PD0332991, versus vehicle (DMSO) for 4 hours
Here, we monitored the early transcriptional response to acute inhibition of CDK4/6 catalytic activity. As PD0332991 substantially induced senescence in U2OS cells, we performed gene expression arrays from U2OS cells treated with or without PD0332991 for 4 hours. Although this did not result in significant changes in the cell cycle profile, it led to significant changes in global gene expression.

overall_design: 10 total samples were analyzed, i.e. we compared five PD0332991-treated versus five vehicle-treated U2OS cell samples (gene level).

first: Peter

last: Sicinski


title: PU.1 restricts adult hematopoietic stem cell proliferation via cell specific autoregulation

city: Singapore

first: Henry

last: Yang

address: 28 Medical Drive

country: Singapore

institute: Cancer Science Institute of Singapore

summary: To guarantee blood supply throughout adult life hematopoietic stem cells (HSCs) need to carefully balance between self-renewing cell divisions and quiescence. Identification of genes controlling HSC self-renewal is of utmost importance given that HSCs are the only stem cells with broad clinical applications. Transcription factor PU.1 is one of the major regulators of myeloid and lymphoid development. Recent reports suggest that PU.1 mediates its functions via gradual expression level changes rather than binary on/off states. So far, this has not been considered in any study of HSCs and thus, PU.1’s role in HSC function has remained largely unclear. Here we demonstrate using hypomorphic mice with an engineered disruption of an autoregulatory feedback loop that decreased PU.1 levels resulted in loss of key HSC functions, all of which could be fully rescued by restoration of proper PU.1 levels via a human PU.1 transgene. Mechanistically, we found excessive HSC cell divisions and altered expression of cell cycle regulators whose promoter regions were bound by PU.1 in normal HSCs. Adequate PU.1 levels were maintained by a mechanism of direct autoregulation restricted to HSCs through a physical interaction of a -14kb enhancer with the proximal promoter. Our findings identify PU.1 as novel regulator controling the switch between cell division and quiescence in order to prevent exhaustion of HSCs. Given that even moderate level changes greatly impact stem cell function, our data suggest important therapeutic implications for leukemic patients with reduced PU.1 levels. Moreover, we provide first proof, that autoregulation of a transcription factor, PU.1, has a crucial function in vivo. We anticipate that our concept of how autoregulation forms an active chromosomal conformation will impact future research on transcription factor networks regulating stem cell fate.

overall_design: HSCs of Pu.1 knock-in (PU.1ki/ki) mice were used for RNA extraction and hybridization on Affymetrix microarrays. We compared these microarray samples with the corresponding wild type.

first: Philipp

last: Staber

first: Pu

last: Zhang

first: Min

last: Ye

first: Robert

last: Welner

first: César

last: Nombela-Arrieta

first: Christian

last: Bach

first: Marc

last: Kerenyi

first: Boris

last: Bartholdy

first: Sanghoon

last: Lee

first: Junyan

first: Mathias

last: Leddin

first: Leslie

last: Silberstein

first: Gerald

last: Höfler

first: Frank

last: Rosenbauer

first: Gang

last: Huang

first: Daniel

last: Tenen


title: Dendritic cell lineage commitment is instructed by distinct cytokine signals

status: Public on Nov 13 2011

city: Aachen

first: Martin

last: Zenke

state: NRW

address: Universitatsklinikum Aachen, RWTH

department: Cell Biology

institute: Institute for Biomedical Engineering

summary: Dendritic cells (DC) develop from hematopoietic stem cells, which is guided by instructive signals through cytokines. DC development progresses from multipotent progenitors (MPP) via common DC progenitors (CDP) into DC. Flt3 ligand (Flt3L) signaling via the Flt3/Stat3 pathway is of pivotal importance for DC development under steady state conditions. Additional factors produced during steady state or inflammation, such as TGF-beta1 or GM-CSF, also influence the differentiation potential of MPP and CDP. Here, we studied how gp130, GM-CSF and TGF-beta1 signaling influence DC lineage commitment from MPP to CDP and further into DC. We observed that activation of gp130 signaling promotes expansion of MPP. Additionally, gp130 signaling inhibited Flt3L-driven DC differentiation, but had little effect on GM-CSF-driven DC development. The inflammatory cytokine GM-CSF induces differentiation of MPP into inflammatory DC and blocks steady state DC development. Global transcriptome analysis revealed a GM-CSF-driven gene expression repertoire that primes MPP for differentiation into inflammatory DC. Finally, TGF-beta1 induces expression of DC-lineage affiliated genes in MPP, including Flt3, Irf-4 and Irf-8. Under inflammatory conditions, however, the effect of TGF- beta1 is altered: Flt3 is not upregulated, indicating that an inflammatory environment inhibits steady state DC development. Altogether, our data indicate that distinct cytokine signals produced during steady state or inflammation have a different outcome on DC lineage commitment and differentiation.

overall_design:
6 samples in total.

Multipotent progenitor
- GM-MPP_1
- GM-MPP_2
Dendritic cell
- GM-DC_1
- GM-DC_2
Dendritic cell plus TNFa
- GM-TNFa-DC_1
- GM-TNFa-DC_2

first: Kristin

last: Sere

first: Qiong

last: Lin

first: Piritta

last: Felker

first: Nina

last: Rehage

first: Theresa

last: Klisch

first: Inga

middle: L

last: Ortseifer

first: Thomas

last: Hieronymus

first: Stefan

last: Rose-John


title: Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts

status: Public on Nov 14 2011

city: Rotterdam

first: Erdogan

last: Taskesen

address: dr. molewaterplein 50

country: Netherlands

institute: Erasmus University Medical Center (EMC)

summary:
Diamond Blackfan Anemia (DBA) is associated with developmental defects and
profound anemia. Mutations in genes encoding a ribosomal protein of the
small (e.g. Rps19) or large (e.g. Rpl11) ribosomal subunit are found in over
half of these patients. The mutations cause   ribosomal haploinsufficiency,
which reduces overall translation efficiency of cellular mRNAs. We reduced
expression of *Rps19* or *Rpl11* in mouse erythroblasts and investigated
mRNA polyribosome association, which revealed deregulated translation
initiation of specific transcripts. Among these were *Bag1*, encoding a
Hsp70 co-chaperone, and *Csde1*, encoding an RNA binding protein, both
expressed at increased levels in erythroblasts. Their translation initiation
is cap-independent and starts from an internal ribosomal entry site (IRES),
which appeared sensitive to knock down of Rps19 or Rpl11. Mouse embryos
lacking Bag1 die at embryonic day E13.5 with reduced erythroid colony
forming cells in the fetal liver, and low Bag1 expression impairs erythroid
differentiation in vitro. Reduced expression of Csde1 impairs proliferation
and differentiation of erythroid blasts. Protein but not mRNA expression of
*BAG1* and *CSDE1* was reduced in erythroblasts cultured from DBA patients.
Our data suggest that impaired IRES-mediated translation of mRNAs expressed
at increased levels in erythroblasts contributes to the erythroid phenotype
of DBA.

overall_design: 3 biological replicates of erythroblasts treated with different shRNA were used for polyribosomal sucrose gradients; RNA was extracted from gradients in 2 samples - mRNA associated with polyribosomes (poly) and the rest (sub).

first: Rastislav

last: Horos

first: Hanna

middle: H

last: Ijspeert

first: Dagmar

last: Pospisilova

first: Andrzej

last: Nieradka

first: Radek

last: Cmejla

first: Ivo

middle: I

last: Touw

first: Marieke

middle: M

last: von Lindern


title: Mitochondrial myopathy elicits global starvation response through FGF21

status: Public on Nov 16 2011

city: Hershey

first: Nuno

last: Raimundo

address: 500 University Dr, College Of Medicine, Room C4769

department: Cellular and Molecular Physiology

institute: Penn State College of Medicine

summary: Progressive mitochondrial respiratory chain (RC) deficiency is associated with a wide spectrum of adult-onset degenerative diseases, as well as with normal aging. We have previously generated the Deletor mice to model late-onset progressive RC defects. Here we report novel tissue-specific pathways contributing to mitochondrial disease pathogenesis, identified by gene expression analysis. We found that RC-deficient muscle fibers secrete the fasting-induced hormone, fibroblast growth factor 21 (FGF21). This response leads to fatty acid recruitment from adipocytes and resistance to high-fat-diet induced obesity in mice, but does not affect glucose or insulin metabolism. FGF21 is also induced in the muscle of mitochondrial myopathy patients and in other RC-deficient mice. These data show that skeletal muscle is an endocrine organ, which signals its energy deficiency through FGF21. Furthermore, RC deficiency in single muscle fibers initiates a global starvation response. These data have important implications for conditions with primary or secondary RC deficiency.

overall_design: Mice overexpressing mutant Twinkle (C10ORF2) protein are the first animal model for Progressive External Ophtalmoplegia (PEO). Using PEO-model and wt-mice, skeletal muscle (quadriceps femoris) was analyzed for gene expression profile.

first: Anu

last: Wartiovaara

first: Henna

last: Tyynismaa


title: Mouse C2C12 cells treated with/without hBMP2

status: Public on Nov 11 2011

city: Shinagawa

first: Atsushi

last: Yamada

state: Tokyo

address: 1-5-8 Hatanodai

department: School of Dentistry

country: Japan

institute: Showa University

summary: Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells.

overall_design: We used cDNA microarrays to elucidate regulators of BMP-2-induced osteoblast differentiation.

first: Dai

last: Suzuki


title: BMP and Activin treatment of mouse extraembryonic endoderm (XEN) cells

status: Public on Nov 10 2011

city: Edinburgh

first: Tilo

last: Kunath

address: 5 LIttle France Drive

department: Centre for Regenerative Medicine

institute: University of Edinburgh

summary:
XEN cells are derived from the primitive endoderm of mouse blastocysts. In culture and in chimeras they exhibit properties of parietal endoderm. However, BMP signaling promotes XEN cells to form an epithelium and differentiate into visceral endoderm (VE). Of the several different subtypes of VE described, BMP induces a subtype that is most similar to the VE adjacent to the trophoblast-derived extraembryonic ectoderm.
The experiment was performed to gain insight into genes regulated by BMP and activin in XEN cells, and also to more precisely define the VE subtypes formed in culture.

overall_design: IM8A1 XEN cells were treated for 6 days with BMP2 (20 ng/ml, R&D Systems), activin A (30 ng/ml, Peprotech), both, or neither in GMEM + 10% fetal bovine serum.

first: Cheryle

last: Sequin

first: Janet

last: Rossant


title: IL-5+ and IL-5- memory Th2

status: Public on Nov 07 2011

city: Chiba-city

first: Yusuke

last: ENDO

state: Chiba

address: 1-8-1 Inohana, Chuo-ku

department: Immunology

institute: Chiba University

summary: We used microarray analysis to identify specific molecular mechanisms controlling IL-5 transcription in memory Th2 cells.

overall_design: IL-5+ and IL-5- memory Th2 cells were selected for RNA extraction and hybridization on Affymetrix microarrays.

last: Endo

first: Chiaki

last: Iwamura

first: Makoto

last: Kuwahara

first: Akane

first: Kaoru

last: Sugaya

first: Damon

last: Tumes

first: Koji

last: Tokoyoda

first: Hiroyuki

last: Hosokawa

first: Masakatsu

last: Yamashita

first: Toshinori

last: Nakayama


title: Expression data from outflow region at E9.5

status: Public on Nov 08 2011

city: Kashihara

first: Masahide

last: Sakabe

address: 840 Shijo-cho

department: Advanced Medical Research Center

institute: Nara Medical University

summary: A high concentration of maternal retinoic acid (RA), the active derivative of vitamin A, is well known as a teratogenic agent which can cause developmental defects. Our previous studies have shown that the maternal administration of RA to mice within a narrow developmental window induces outflow tract (OFT) septum defects, which closely resembles human transposition of the great arteries (TGA), although the responsible factors and pathogenic mechanisms of the TGA induced by RA still remain unknown. To identify the candidate genes involved in the altered development of the OFT in RA-induce TGA model, we isolated the RNA from E9.5 control and RA-treated OFT regions, and performed a microarray analysis.

overall_design: Approximately 200 outflow tract region were isolated from control and RA-treated embryos (embryonic day 9.5). We sought to obtain the candidate genes involved in the altered development of the OFT in RA-induce heart defects.

first: Yuji

last: Nakajima

first: Yumiko

last: Saga


title: Differential expression of genes in pancreatic islet of C57Bl/6J on high fat diet

status: Public on Nov 20 2011

city: Norfolk

first: yumi

last: imai

state: VA

address: 799W Olney Road, LH 2156

institute: Eastern Virginia Medical School

summary: Male C57Bl/6J mice were fed 45%kcal fat diet (HF) or regular rodent chow (NC) from 4 weeks to 16 weeks of age. Gene expression was compared between RNA obtained from pancreatic islets of HF fed mice and NC mice.

overall_design: RNA samples from 4 NC group and 4 HF groups were analyzed using GeneChip Mouse Expression Arrays MOE 430v2 (Affymetrix).

first: Yumi

last: Imai

first: Rexford

last: Ahima


title: Expression data from C/EBP alpha induced transdifferentiation of pre-B cells into macrophages

city: marseille

first: Thien-Phong

last: Vu Manh

address: 163 avenue de Luminy

country: France

institute: CIML

summary:
Earlier work has shown that pre-B cells can be converted into macrophages by the transcription factor C/EBP? at very high frequencies. Using this system we have now performed a systematic analysis of the question whether during transdifferentiation the cells transiently reactivate progenitor restricted genes or even retrodifferentiate. A transcriptome analysis of transdifferentiating cells showed that most genes are continuously up or downregulated, acquiring a macrophage phenotype within 5 days. In addition, we observed the transient reactivation of a subset of immature myeloid markers, as well as low levels of the progenitor markers Kit and Flt3 and a few lineage inappropriate genes. However, we were unable to observe the re-expression of cell surface marker combinations that characterize hematopoietic stem and progenitor cells (HSPCs), including c-Kit and Flt3. This was the case even when C/EBPalpha was activated in pre-B cells under culture conditions that favor HSPC growth or when the transcription factor was activated in a time limited fashion. Together, our findings are consistent with the notion that the conversion from pre-B cells to macrophages is mostly direct and does not involve overt retrodifferentiation.

Our microarray data indicate that most expression changes are direct, and that transdifferentiation does not involve retrodifferentiation. Our data also show that there is a low level transient activation of selected progenitor genes, as well as myeloid precursor genes.

overall_design: Primary mouse pre-B cells were infected with an inducible form of C/EBPa and cells induced to transdifferentiate with beta Estradiol (b-Est) harvested at different time points to analyze expression profiles, using Affymetrix 430.2 arrays.

first: A

last: Di Tullio

first: T

last: Schubert

first: R

last: Mansson

last: Graf


title: Circadian temporal profiling of MMH-D3 hepatocytes

status: Public on Nov 03 2011

first: Susanna

middle: S

last: Wang

address: 9500 Gilman Dr.

department: Biological Sciences

summary: The circadian clock generates daily rhythms in mammalian liver processes, such as glucose and lipid homeostasis, xenobiotic metabolism, and regeneration. The mechanisms governing these rhythms are not well understood, particularly the distinct contributions of the cell-autonomous clock and central pacemaker to rhythmic liver physiology. Through microarray expression profiling in MMH-D3 hepatocytes, we identified over 1,000 transcripts that exhibit circadian oscillations, demonstrating that many rhythms can be driven by the cell-autonomous clock and that MMH-D3 is a valid circadian model system. The genes represented by these circadian transcripts displayed both co-phasic and anti-phasic organization within a protein-protein interaction network, suggesting the existence of competition for binding sites or partners by genes of disparate transcriptional phases. Multiple pathways displayed enrichment in MMH-D3 circadian transcripts, including the polyamine synthesis module of the glutathione metabolic pathway. The polyamine synthesis module, which is highly associated with cell proliferation and whose products are required for initiation of liver regeneration, includes enzymes whose transcripts exhibit circadian oscillations, such as ornithine decarboxylase (Odc1) and spermidine synthase (Srm). Metabolic profiling revealed that the enzymatic product of SRM, spermidine, cycles as well. Thus, the cell-autonomous hepatocyte clock can drive a significant amount of transcriptional rhythms and orchestrate physiologically relevant modules such as polyamine synthesis.

overall_design: Samples were collected every 2 hours for a duration of 46 hours from differentiated MMH-D3 hepatocytes synchronized via serum shock. Cells were synchronized by serum shock and incubated for 12 hours, and then samples were collected every 2 hours from 12 hours post-serum shock to 58 hours post-serum shock, for a total of 24 samples.

first: Ann

last: Atwood

first: Steve

last: Kay


title: mRNA expression data of tumor samples with/without IDH1/2 mutations and of controls

status: Public on Nov 05 2011

city: Leiden

first: Twinkal

middle: Chandubhai

last: Pansuriya

state: Zuid-Holland

address: Albinusdreef 2

department: Pathology

institute: LUMC

summary: We determined differentially expressed genes between tumor samples with and without IDH1/2 mutations, and between tumors with IDH1/2 mutations and controls.

overall_design: A total of 21 tumours which include 6 enchondromas and 10 chondrosarcomas (3 grade I, 7 grade II) of Ollier disease, as well as 1 enchondroma and 4 chondrosarcomas grade II of solitary tumors, and 6 controls (growth plate and cartilage) were selected for expression analysis. Sample preparation, RNA isolation, synthesis of cDNA, cRNA amplification, hybridization of cRNA onto the Illumina Human-6 v3.0 Expression BeadChips, microarray data preprocessing, quality control, and detection of differential expression were performed as described previously (PMID: 21372215, PMID: 21584901). We determined differential expression between tumor samples without detectable IDH1 or IDH2 mutation (n=3) and samples with IDH1 or IDH2 mutations (n=18), and between samples with IDH1 or IDH2 mutation and controls (n=6). Probes with Benjamini and Hochberg False discovery rate-adjusted P-values (adjP) < 0.05 and a log fold change (logFC) > 0.1 were considered to be significantly differentially expressed.

middle: C

first: Judith

last: Bovee

first: Jan

last: Oosting

first: Marieke

last: Kuijjer


title: Somatic mosaic IDH1 and IDH2 mutations are associated with enchondroma and spindle cell haemangioma in Ollier disease and Maffucci syndrome

type: Methylation profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection (stem cell clones NESC,TASC,DASC)

status: Public on Nov 01 2011

first: Frank

middle: Daniel

last: McKeon

address: 60 Biopolis street

department: Stem cell and Developmental Biology 7

institute: Genome Institute of Singapore

summary: The extent of lung regeneration following catastrophic damage and the potential role of adult stem cells in such a process remains obscure.  Sublethal infection of mice with an H1N1 influenza virus related to that of the 1918 pandemic triggers massive airway damage followed by apparent regeneration.  We show here that p63-expressing stem cells in the bronchiolar epithelium undergo rapid proliferation after infection and radiate to interbronchiolar regions of alveolar ablation.  Once there, these cells assemble into discrete, Krt5+ pods and initiate expression of markers typical of alveoli.  Gene expression profiles of these pods suggest that they are intermediates in the reconstitution of the alveolar-capillary network eradicated by viral infection.  The dynamics of this p63-expressing stem cell in lung regeneration mirrors our parallel findings that defined pedigrees of human distal airway stem cells assemble alveoli-like structures in vitro and suggests new therapeutic avenues to acute and chronic airway disease.

overall_design: The long-term self-renewal potential of the putative human stem cell clones (NESC,TASC,DASC) allowed us to isolate independent pedigrees for the analysis of the progeny of a single cell. Expansion of these lines yielded abundant immature cells of known pedigree for a range of differentiation assays to assess lineage potential. All stem cells were grown on feeder layer (3T3).NESC were differentiated using Air liquid interface system (ALI), 3D matrigel system (MAT) and Self assembling sphere culture(SAS). The differentiation potential of TASC and DASC were studied using ALI and MAT systems.

first: Pooja

last: Kumar

first: Yuanyu

last: Hu

first: Yusuke

last: Yamamoto

first: Neo

last: Hoe

first: Tay

last: Wei

first: Dakai

last: Mu

first: Yan

last: Sun

first: Lim

last: Joo

first: Rania

last: Daghers

first: Elisabeth

last: Zielonka

first: De

middle: Y

first: Vincent

middle: T

last: Chow

first: Christopher

middle: P

last: Crum

first: Wa

last: Xian


title: Discrete subsets of myogenic and mesenchymal precursors give rise to soft tissue sarcomas of distinct lineage differentiation

first: Amy

last: Wagers

address: 1 Joslin Place

department: Islet Cell and Regenerative Biology

institute: Joslin Diabetes Center

summary: This work examines sarcoma formation within discrete subsets of KRAS(G12V)-expressing p16p19null myogenic and mesenchymal cells found normally in skeletal muscle. We show that prospectively isolated skeletal muscle precursor cells (SMPs) within the satellite cell pool can serve as cancer cells-of-origin for mouse rhabdomyosarcomas (soft tissue sarcomas with features of myogenic differentiation). Alternatively, non-myogenic progenitors (ScaPCs) induce sarcomas lacking myogenic differentiation markers.

overall_design: We used Affymetrix whole mouse genome 430 2.0 microarrays to gain deeper insights into the molecular underpinnings of the three types of KRAS; p16p19null mouse soft-tissue sarcomas (originating from SMPs, ScaPCs and CD45-MAC1-TER119-Sca1-CXCR4- cells). Four replicates of each type.

first: Simone

last: Hettmer

first: Jianing

last: Liu

first: Roderick

last: Bronson

first: David

last: Langenau


title: Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection

first: Frank

last: McKeon

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Microarray expression analysis in idiopathic and LRRK2-associated Parkinson's disease

city: Barcelona

first: Teresa

last: Botta

address: Casanova 143

department: Serveis Científicotècnics

institute: IDIBAPS-Universitat de Barcelona-Hospital Clínic

summary: LRRK2 mutations are the most common genetic cause of Parkinson’s disease (PD). We performed a whole-genome RNA profiling of putamen tissue from idiopathic PD (IPD), LRRK2-associated PD (G2019S mutation), neurologically healthy controls and one asymptomatic LRRK2 mutation carrier, by using the Genechip Human Exon 1.0-ST Array. The differentially expressed genes found in IPD revealed an alteration of biological pathways related to long term potentiation (LTP), GABA receptor signalling, and calcium signalling pathways, among others. These pathways are mainly related with cell signalling cascades and synaptic plasticity processes. They were also altered in the asymptomatic LRRK2 mutation carrier but not in the LRRK2-associated PD group. The expression changes seen in IPD might be attributed to an adaptive consequence of a dysfunction in the dopamine transmission. The lack of these altered molecular pathways in LRRK2-associated PD patients suggests that these cases could show a different molecular response to dopamine transmission impairment.

overall_design: Number of samples analyzed: 14 brain tissue (putamen). Control (C) samples, no pathological changes: C1, C2, C3, C4, C5. Samples from idiopathic Parkinson's Disease (IPD) patients: IPD1, IPD2, IPD3, IPD4, IPD5. Samples from Parkinson's disease patients carriers of G2019S LRRK2 mutation (MPD): MPD1, MPD2, MPD3. Sample from an asymptomatic carrier of G2019S LRRK2 mutation (MC). No replicates.

last: Botta-Orfila

first: Eduardo

last: Tolosa

first: Ellen

last: Gelpi

first: Alex

last: Sànchez-Pla

first: María-José

last: Martí

first: Francesc

last: Valldeoriola

first: Manel

last: Fernández

last: Carmona

first: Mario

last: Ezquerra


title: Gene expression atlas for mouse olfactory sensory neurons

status: Public on Nov 02 2011

city: Lexington

first: Tim

last: McClintock

state: KY

address: 780 Rose St.

department: Physiology

institute: University of Kentucky

summary: Identification of all genes expressed by mouse olfactory sensory neurons; genes expressed in mature neurons, immature neurons, or both were distinguished.  Independent validation of enrichment ratio values supported by statistical assessment of error rates was used to build a database of statistical probabilities of the expression of all mRNAs detected in mature neurons, immature neurons, both types of neurons (shared), and the residual population of all other cell types.

overall_design: FACS was used to separate a highly GFP+++ sample enriched in immature neurons, a weakly GFP+ sample slightly enriched in mature neurons, and a GFP- sample that contained all cell types in the olfactory epithelium but depleted of immature neurons.

first: Melissa

middle: D

last: Nickell

first: Patrick

last: Breheny

first: Arnold

last: Stromberg

first: Timothy


title: Expression profiling of erythroid developmental subsets isolated from mouse fetal liver.

status: Public on Oct 31 2011

city: Worcester

first: Merav

last: Socolovsky

address: 364 Plantation Street

department: Pediatrics and Cancer Biology

institute: University of Massachusetts Medical School

summary:
The S1 and S3 erythroid developmental subsets were isolated using flow cytometry and the cell surface markers CD71 and Ter119 as described by Pop et. al. 2010 (PMID: 20877475)
Expression profiles for S1 and S3 subsets were generated using Affymetrix GeneChips.  Results were used to identify genes that are differentially expressed during erythropoiesis.

overall_design: Single cell suspensions were prepared by mechanically dissociating whole fetal livers obtained from E12.5 to E13.5 Balb/C mouse embryos. Cells were stained  for CD71, Ter119, and a cocktail containing lineage-specific antibodies. S1 and S3 erythroid developmental subsets were identified and isolated using flow cytometric sorting as described by Pop et. al. 2010 (PMID: 20877475). S1 and S3 subsets were isolated on 3 seperate days to generate total RNA (biological replicates).  20 ng of total RNA from each biological replicate was converted to cDNA, linearly amplified and biotinylated using Ovation reagents (Nugen, San Carlos, CA).  Samples were hybridized to Mouse  Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA). Microarray suite 5 (MAS5) processed sample data were normalized to the average of 18SRNA (AFFX-18SRNAMur/X00686_M_at), GAPDH (AFFX-GapdhMur/M32599_3_at) and β-actin (1419734_at) expression values. These gene expression profiles were performed as part of the manuscript by Shearstone et. al. Global DNA Demethylation During Erythropoiesis In Vivo

first: Jeffrey

middle: R

last: Shearstone


title: Expression data from mouse embryonic stem cells lacking the Alkbh1 gene

city: Oslo

first: Rune

last: Ougland

address: Sognsvannsveien 20

department: Dept. of Microbiology

country: Norway

institute: Oslo University Hospital, Rikshospitalet

summary: AlkB homolog 1 (ALKBH1) is one of nine members of the AlkB homologs in mammals. Most Alkbh1-deficient mice die during embryonic development, and survivors have severe defects in tissues originating from the ectodermal lineage. We hypothesized the phenotype to rely upon aberrant epigenetic regulation and provided evidence for ALKBH1 to be a histone H2A demethylase. We used a whole genome expression microarray to detail differentially expressed genes in embryonic stem cells lacking the Alkbh1 gene and identified distinct classes of up- and down-regulated genes during this process.

overall_design: Wild type and Alkbh-/- embryonic stem cells were established to identify differentially expressed genes at day 0, undifferentiated cells. We sought to obtain a subset of genes possibly explaining the observed phenotype. Total RNA was extracted and hybridized on Affymetrix GeneChips. 3 parallels were analyzed from each of the two genotypes.

first: Elisabeth

last: Larsen


title: Transcriptional Profile of Aging in Healthy Human Skin

city: Perth

first: Daniel

middle: J

last: Haustead

state: WA

address: 35 stirling highway (M317)

department: Surgery

institute: University Of Western Australia

summary: Gene expression changes were assessed from the non sun-exposed skin of the lower back of 98 healthy males aged 19-86. We show that contrary to previous thought, genome wide transcriptional activity does not display an exclusively linear correlation with ageing, but rather, in human skin, undergoes a period of significant transient change between 30 and 45 years of age. The identified transient transcriptional changes suggest a period of heightened metabolic activity and cellular damage mediated primarily through the actions of TP53 (tumour protein 53) and TNF (tumour necrosis factor). We also identified a subgroup of the population characterised by increased expression of a large group of hair follicle genes that correlates strongly with a younger age of onset and increasing severity of androgenetic alopecia.

overall_design: Skin was collected from the lower back at the level of the belt, aproximately 5cm lateral to midline from healthy males, (defined as; non-smoking, no hospital admissions in the previous 5 years, no significant medical conditions or medications). Each sample was individually hybridised to an exon 1.0 ST array.

first: Mark

last: Fear


title: Nuclear receptors Nur77 and Nurr1 modulate mesenchymal stromal cell migration

status: Public on Oct 24 2011

city: Amsterdam

first: Marijke

last: Maijenburg

address: Plesmanlaan 125

department: Experimental Immunohematology

institute: Sanquin Research

summary:
Expression analysis of migrating and non-migrating mesenchymal stromal cells (MSC) in fetal bone marrow

Keywords: fetal bone marrow, mesenchymal stromal cells, migration, gene expression, genomics

overall_design: Three biological replates for both migrating and non-migrating mesenchymal stromal cells (MSC) in fetal bone marrow

first: Christian

last: Gilissen

first: Sara

last: Melief

first: Marion

last: Kleijer

first: Kees

last: Weijer

first: Anja

last: ten Brinke

first: Helene

last: Roelofs

first: Claudia

last: van Tiel

first: Joris

last: Veltman

first: Carlie

last: de Vries

first: Ellen

last: van der Schoot

first: Carlijn

last: Voermans


title: Transcriptional profile of ER71 EYFP positive/negative cells in ER71 WT and MT embryos

city: Minneapolis

first: Fengli

last: Fu

state: MN

address: 312 Church Street SE

institute: University of Minnesota

summary: ER71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages.   To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models.  We engineered an ER71-EYFP transgenic mouse model by fusing the 3.9 kb ER71 promoter to the EYFP reporter gene.  Using FACS and transcriptional profiling, we examined the EYFP+ populations of cells in ER71 mutant and wildtype littermates.  In the absence of ER71, we observed an increase in the number of EYFP expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program compared to the wildtype littermate controls.  We have also generated a novel ER71-Cre transgenic mouse model using the same 3.9 kb ER71 promoter.  Genetic fate mapping studies revealed that the ER71 expressing cells daughter hematopoietic and endothelial lineages in the wildtype background.  In the absence of ER71, these cell populations contributed to alternative mesodermal lineages including the cardiac lineage.  To extend these analyses, we used an inducible ES/EB system and observed that ER71 overexpression repressed cardiogenesis.  Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions, acting to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages.  This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.

overall_design: 12samples were analyzed, including triplicates of WT; EYFP positive, WT EYFP negative, ER71 MT; EYFP positive and ER71 MT; EYFP negative cells

first: D

last: Garry

first: T

last: Rasmussen


title: Comparative Transcriptional Network Modeling of Three PPAR-α/γ Co-agonists Reveals Distinct Metabolic Gene Signatures in Primary Human Hepatocytes

status: Public on Oct 22 2011

city: Basel

first: Guido

last: Steiner

address: Grenzacher Strasse

department: TRS/BEDA - Biostatistics

institute: Hoffmann La-Roche

summary: Aims: To compare the molecular and biologic signatures of a balanced dual peroxisome proliferator-activated receptor-α/γ (PPAR-α/γ) agonist, aleglitazar, with tesaglitazar (a dual PPAR-α/γ agonist) or combination of pioglitazone (PPAR-γ agonist)/fenofibric acid (PPAR-α agonist) in human hepatocytes. Methods and Results: Gene expression microarray profiles were obtained from primary human hepatocytes treated with EC50-aligned low, medium, and high concentrations of the three treatments. A systems-biology approach, Causal Network Modeling, was used to model the data to infer upstream molecular mechanisms that may explain the observed changes in gene expression. Aleglitazar, tesaglitazar and pioglitazone/fenofibric acid, each induced unique transcriptional signatures, despite comparable core PPAR signaling. Although all treatments inferred qualitatively similar PPAR-α signaling, aleglitazar was inferred to have greater effects on high- and low-density lipoprotein cholesterol levels than tesaglitazar and pioglitazone/fenofibric acid, due to greater number of gene expression changes in pathways related to HDL and LDL metabolism. Distinct transcriptional and biologic signatures were also inferred for stress responses, which appeared to be less affected by aleglitazar than the comparators. In particular, pioglitazone/fenofibric acid was inferred to increase NFE2L2 activity, a key component of the stress response pathway, while aleglitazar had no significant effect. All treatments were inferred to decrease proliferative signaling. Conclusions: Aleglitazar induces transcriptional signatures related to lipid parameters and stress responses that are unique from other dual PPAR-α/γ treatments. This may underlie observed favorable changes in lipid profiles in animal and clinical studies with aleglitazar and suggests a differentiated gene profile compared with other dual PPAR-α/γ agonist treatments.

first: Renée

last: Kenney

first: Pia

last: Maerz-Weiss

first: Natalie

last: Catlett

first: Ben

last: Wong

first: Matthew

last: Wrigh

first: Gil

last: Blander

first: Keith

middle: O

last: Elliston

first: William

last: Ladd

first: Maria

last: Bobadilla

first: Jacques

last: Mizrahi

first: Carolina

last: Haefliger

first: Alan

last: Edgar


title: Expression data from purified control and BrdtΔBD1 round spermatids.

status: Public on Oct 21 2011

city: New York

first: Binyamin

middle: David

last: Berkovits

state: NY

address: 1130 St. Nicholas Ave. Room 611

department: Genetics and Development

institute: Columbia University

summary:
Brdt is a testis specific member of a family of chromatin interacting proteins. All of the family members have been shown to regulate transcription. Brdt is highly expressed in round spermatids, and may play a role in transcriptional regulation in these cells.
We investigated transcriptional changes in mutant round spermatids that were homozygous for a mutation in which the first bromodomain of Brdt was removed.

overall_design: Round spermatids were purified from seven adult animals of each genotype for each Affymetrix microarray. Purity of round spermatids was assessed by propidium iodide staining.

first: Paolo

last: Guarnieri

first: Debra

last: Wolgemuth


title: The immunoregulatory effect of macrophage-specific PPAR gamma deficiency on experimental inflammatory bowel disease

status: Public on Nov 12 2011

city: Baltimore

last: Salvato

state: MD

address: 725 W Lombard Street

department: Institute of Human Virology

institute: University of Maryland School of Medicine

summary: Inflammatory bowel disease (IBD) is a condition characterized by severe intestinal inflammation and immune cell activation.  The severity of the disease can be mitigated by compounds which activate peroxisome proliferator-activated receptor gamma (PPAR gamma), a receptor present widely in tissues involved in IBD pathogenesis.  Our objective was to assess the affect of macrophage-specific deficiency of PPAR gamma on peripheral and colonic immune populations and colonic gene expression in experimental IBD.  Macrophage-specific PPAR gamma-deficient mice (PPAR gamma flfl Lysozyme M Cre+) and control (PPAR gamma flfl Lysozyme M Cre-) littermates were treated with 2.5% dextran sodium sulfate (DSS) for 7 days.  Disease activity was recorded daily and immune cell populations in the blood, spleen, mesenteric lymph nodes (MLN), and lamina propria were examined by flow cytometry.  Colonic gene expression was assessed by real time PCR and microarray analyses.  Our findings show that macrophage PPAR r-deficiency significantly exacerbates DSS inflammation.   CD4+CD25+FoxP3+ regulatory T cells (T-regs) were significantly reduced in Cre+ mice, and MLN macrophages and CD40 expression were enhanced.  There were significant differences in the number colonic macrophages between Cre+ and Cre- mice, but those from Cre+ mice expressed more CD40, Ly6C, and TLR-4.  PPAR r-deficiency also increased the percent of CD8+ T cells in the lamina propria and enhanced colonic interferon gamma expression.  Our findings indicate that macrophage PPAR gamma deficiency augments the severity of DSS colitis by reducing peripheral T-regs and increasing colonic macrophage activation and T cell inflammation.

overall_design: RNA from 3 PPAR gamma-deficient mice (PPAR gamma flfl; Lysozyme M Cre+) and 3 control (PPAR gamma flfl; Lysozyme M Cre-) littermates was processed and labeled according to the standard target labeling protocols. The samples were hybridized, stained, and scanned per standard Affymetrix protocols at the Virginia Bioinformatics Institute (VBI) core laboratory on Mouse 430 2.0 expression arrays (Affymetrix Inc., Santa Clara, CA).

first: Raquel

last: Hontecillas

last: Horne

first: Amir

last: Guri

first: Yan

last: Zhang

first: Bruno

last: Sobral

first: Josep

last: Bassaganya-Riera


title: Distinct Protein Degradation Induced by Different Disuse Models of Skeletal Muscle Atrophy

status: Public on Oct 25 2011

city: Cambridge

first: Scott

last: Jelinsky

state: MA

address: 610 Main Street

department: Inflammation and Immunology

institute: Pfizer

summary: Skeletal muscle atrophy is a consequence of many diseases, environmental insults, inactivity, age and injury.  Atrophy is characterized by active degradation and removal of contractile proteins and a reduction in fiber size.  Animal models have been extensively used to identify pathways leading to atrophic conditions.  Here we have used genome-wide expression profiling analysis and quantitative PCR to identify the molecular changes that occur in two clinically relevant animal mouse models of muscle atrophy, hindlimb casting and Achilles tendon laceration (tenotomy).  Gastrocnemius muscle samples were collected 2, 7 and 14 days after insult.  The total amount of muscle loss as measured by wet weight and muscle fiber size was equivalent between models, although tenotomy resulted in a more rapid induction of muscle atrophy.  Furthermore, tentomy resulted in the regulation of significantly more mRNA transcripts then casting.  Analysis of the regulated genes and pathways suggest that the mechanism of atrophy is distinct between these models.  The degradation following casting appears ubiquitin-proteasome-mediated while degradation following tenotomy appears lysosomal and matrix-metalloproteinase (MMP)-mediated.  This data suggests that there are multiple mechanisms leading to muscle atrophy and that specific therapeutic agents may be necessary to combat the atrophy seen under different conditions.

overall_design: Muscle atrophy was induced through two methods; unloading induced by tendon laceration (Tenotomy) and immobilization induced by hindlimb casting (Casting).  For the tenotomy, eight week old male C57/B6 mice were anesthetized by ether inhalation and subsequent intramuscular injection of ketamine-xylazine (0.1 ml/g). The right Achilles tendon just proximal to the calcaneus was severed with a sterile scalpel.  The mice were allowed to recover and move freely about their cage.  An additional cohort of animals was sham operated (Sham) which involved isolation and manipulation of the Achilles tendon without laceration.  For the casting, the right hindlimbs of eight week old male C57Bl/6 mice were immobilized through casting.  After 2, 7 or 14 days, animals were sacrificed and the left and right gastrocnemius muscles were carefully removed, flash frozen in liquid nitrogen, and stored at -80°C for further processing.  The left untreated legs served as controls.  A group of naïve animals that received no treatment or manipulation was included as an additional control.  Each group consisted of between 5 and 10 samples for a total of 111 individual samples.


title: Synergism between curdlan and GM-CSF in mouse dendritic cells

status: Public on Oct 15 2011

city: Singapore

first: Michael

last: Poidinger

address: #04-06, 8A Biomedical Grove

department: Bioinformatics

institute: Singapore Immunology Network

summary: A simultaneous engagement of different pathogen recognition receptors provides a tailor made adaptive immunity for an efficient defence against distinct pathogens. For example, cross talk of TLR and c-type lectin signalling effectively shapes distinct gene expression patterns by integrating the signals at the level of NF-κB. Here, we extend this principle to a strong synergism between the Dectin-1 agonist, curdlan, and an inflammatory growth factor, GM-CSF. Both together act in synergy in inducing a strong inflammatory signature which converts immature DCs to potent effector DCs. A variety of cytokines (IL-1β, IL-6, TNF-α, IL-2 and IL-12p70), costimulatory molecules (CD80, CD86, CD40 and CD70), chemokines  (CxCl1, CxCl2, CxCl3, CCl12, CCl17) as well as receptors and molecules involved in fugal recognition and immunity such as Mincle, Dectin-1, Dectin-2 and Pentraxin 3 are strongly up-regulated in DC treated simultaneously with curdlan and GM-CSF. The synergistic effect of both stimuli resulted in strong IKBα phosphorylation, in its rapid degradation and in enhanced nuclear translocation of all NF-κB subunits. We further identified MAPK ERK, as one possible integration site of both signals, since its phosphorylation was clearly augmented when curdlan was co-applied with GM-CSF. Our data demonstrate that the immunomodulatory activity of curdlan requires an additional signal provided by GM-CSF to successfully initiate a robust β-glucan specific cytokine and chemokine response. The integration of both signals clearly prime and tailor a more effective innate and adaptive response against invading microbes and fungi.

overall_design: CD11b+ fraction of FLT3L generated BM DCs (3-4 x106) were stimulated for 4 hours with 100 or 1 μg/ml curdlan in presence or absence of 5 ng/ml GM-CSF in triplicates.

first: Christiane

last: Ruedl

first: Alessandra

last: Mortellaro

first: Ottavio

last: Beretta


title: Cell type specific expression data from Mecp2 null mice

city: Ashburn

first: Ken

last: Sugino

address: 19700 Helix Dr

institute: Janelia Research Campus

summary: Mutations in methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome and related autism spectrum disorders. MeCP2 is believed to be required for proper regulation of brain gene expression, but prior microarray studies in Mecp2 knockout mice using brain tissue homogenates have revealed only subtle changes in gene expression. Here, by profiling discrete subtypes of neurons we uncovered more dramatic effects of MeCP2 on gene expression, overcoming the "dilution problem" associated with assaying homogenates of complex tissues. The results reveal misregulation of genes involved in neuronal connectivity and communication. Importantly, genes up-regulated following loss of MeCP2 are biased toward longer genes but this is not true for down-regulated genes, suggesting MeCP2 may selectively repress long genes. Since genes involved in neuronal connectivity and communication, such as cell adhesion and cell-cell signaling genes, are enriched among longer genes, their misregulation following loss of MeCP2 suggests a possible etiology for altered circuit function in Rett syndrome.

overall_design: Transgenic mice lines which label subpopulations of neurons (G42: fast spiking Parvarbumin positive interneurons, YFPH: layer 5 thick tufted pyramidal neurons, TH: tyrosine hydroxylase positive locus coeruleus neurons) were used to obtain cell type specific expression profiles on Affymetrix microarrays. Females which carry Mecp2 null alleles (and one of the fluorescent alleles) were crossed with males (which may or may not carry one of the fluorescent alleles depending on whether the female has one or not). Male offsprings at ages P37 to P55 days which carry fluorescent allele and Mecp2 null allele were used for experiments for post-symptomatic condition and ages P22 to P25 days were used for pre-symptomatic condition (TH_LCY). Littermate males which carry fluorescent allele but not Mecp2 null allele were used for controls. 3 or 4 biological replicates were done for each condition.

first: Chris

last: Hempel

first: Hannah

last: Arnson

first: Sacha

middle: B

last: Nelson

first: Ben

last: Okaty

first: Saori

last: Kato


title: Comparative gene expression analysis of repro9/repro9 and wild type testes from 14 and 17 day mice

status: Public on Oct 19 2011

city: Ithaca

first: John

last: Schimenti

department: Biomedical Sciences

institute: Cornell University

summary:
Repro9 in an allele of Mybl1 (A-Myb) transcription factor obtained in ENU screen to identify alleles causing mouse infertility. Repro9/repro9 mutant males are infertile due to meiotic arrest at pachytene stage. Mutants show wide range of abnormalities including inefficient chromosome synapsis, sex body formation and progression through meiotic cycle. Females are unaffected. To determine genes transcriptionally regulated by MYBL1 we analyzed gene expression profiles of wild type and repro9/repro9 mutant testis at 14 and 17 days postpartum. Analysis revealed many misregulated genes, in majority downregulated, at day 14 pp and even more at day 17 pp, probably due to secondary effects of meiotic arrest. Significantly misregulated genes were characterized by Gene Ontology. Comparative gene expression analysis uncovered potential targets of MYBL1 regulation that play roles in regulation of transcription, cell cycle, apoptosis, protein phosphorylation and ubiquitination, chromosome organization and others.
Abstract:The transcriptional regulation of mammalian meiosis is poorly characterized, due to few genetic and ex vivo models.  From a genetic screen, we identify the transcription factor MYBL1 as a male-specific master regulator of several critical meiotic processes.  Spermatocytes bearing a novel separation-of-function allele (Mybl1repro9) had subtle defects in autosome synapsis in pachynema, a high incidence of unsynapsed sex chromosomes, incomplete double strand break (DSB) repair on synapsed pachytene chromosomes, and a lack of crossing-over.  MYBL1 protein appears in pachynema, and its mutation caused specific alterations in expression of diverse genes, including some translated postmeiotically.  These data, coupled with chromatin immunoprecipitation (ChIP-chip) experiments and bioinformatic analysis of promoters, identified direct targets of MYBL1 regulation.  Meiosis in mutant females appears unaffected. These results reveal that MYBL1 is a master regulator of meiotic genes that are involved in multiple processes in spermatocytes, including chromosome synapsis, recombination, and cell cycle progression through pachynema.

overall_design: RNA was extracted from  wt and repro9/repro9 mutant testis from 14 and 17 days old mice. This two time points were selected for sample enrichment in early/midpachytene and latepachytene/diplotene spermatocytes, respectively. Total testis RNA from wild-type (≥3) and mutant (≥3) mice was reverse-transcribed into double stranded cDNA, and biotin-labeled cRNA (GeneChip IVT labeling kit, Affymetrix, Santa Clara, CA) was hybridized to Affymetrix mouse genome 430 2.0 GeneChips containing.  The raw data was processed using Affymetrix GCOS software. Two-way ANOVA analysis resolved differentially expressed (DE) genes either between genotypes and/or days using MeV(version 4.6.1) on log2-transformed expression values.  Results were multiple test corrected with the Benjamini-Hochberg method to control the false discovery rate (FDR) in R. ~800 genes significant at FDR<=0.025 were selected as differentially expressed for downstream analysis.

first: Ewelina

last: Bolcun-Filas


title: Cyclin D1, Id1 and EMT in breast cancer

status: Public on Oct 13 2011

city: Carrington Crescent

first: Andrew

last: Sims

state: Edinburgh

address: Systems Medicine Building

department: Institute of Genetics and Molecular Medicine

institute: University of Edinburgh

summary: Cyclin D1 is a well characterised cell cycle regulator with established oncogenic capabilities. Despite these properties, studies report contrasting links to tumour aggressiveness. It has previously been shown that silencing cyclin D1 increases the migratory capacity of MDA-MB-231 breast cancer cells with concomitant increase in ‘inhibitor of differentiation 1’ (ID1) gene expression. Id1 is known to be associated with more invasive features of cancer and with the epithelial-mesenchymal transition (EMT). Here, we sought to determine if the increase in cell motility following cyclin D1 silencing was mediated by Id1 and enhanced EMT-features. To further substantiate these findings we aimed to delineate the link between CCND1, ID1 and EMT, as well as clinical properties in primary breast cancer. The increase in cell migration following cyclin D1 silencing in MDA-MB-231 cells was abolished by Id1 siRNA treatment and we observed cyclin D1 occupancy of the Id1 promoter region. Moreover, ID1 and SNAI2 gene expression was increased following cyclin D1 knock-down, an effect reversed with Id1 siRNA treatment. Similar migratory and SNAI2 increases were noted for the ER-positive ZR75-1 cell line, but in an Id1 independent manner. In a meta-analysis of 1107 breast cancer samples, CCND1 and ID1 gene expression were associated with mesenchymal-markers including SNAI1, SNAI2 and TWIST1, and with clinicopathological parameters. Finally, a greater percentage of CCND1low/ID1high tumours were found in the EMT-like ‘claudin-low’ subtype of breast cancer than in other subtypes. Together, these results indicate that increased migration of MDA-MB-231 cells following cyclin D1 silencing can be mediated by Id1 and is linked to an increase in EMT markers. Moreover, we have confirmed a relationship between cyclin D1, Id1 and EMT in primary breast cancer, supporting our in vitro findings that low cyclin D1 expression can be linked to aggressive features in subgroups of breast cancer.

overall_design: MDA-MB-231 cells were transfected with cyclin D1, CDK4/6 or control siRNA.

first: Nicolas

last: Tobin

first: Katja

middle: L

last: Lundgren

first: Göran

last: Landberg