Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification of stage-specific gene expression signatures in response to retinoic acid during the neural differentiation of mouse embryonic stem cells

ABSTRACT: We analyzed gene expression profiling by an bioinformatic approach to identify the stage-specific gene sets associated with neural differentiation of mESCs. We exposed mESCs (B6G-2 cells) to titrated doses of retinoic acid (RA) for 4 days during embryoid body formation and then performed morphological analysis on day of differentiation (DoD) 8 and 29, or genomic microarray analysis on DoD 0, 2, 8 and 29. Microarray data were analyzed by clustering methods such as self-organizing map and principal component analysis. A gene network analysis for selected gene sets was then performed using Bayesian statistics to identify stage-specific gene expression signatures in response to RA. 9 total samples: 1 vehcle, 8 Retinoic acid and two or three doses of each developmental stage(Day) [Vehicle Control (0.1% dimethyl sulfoxide), estradiol (1nM and 10nM), dehydrotestosterone (1nM and 10nM) bisphenol A (1pM and 100pM), triiodothyronine (1nM and 10nM), dexamethasone (1nM and 10nM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (1nM and 10nM), 4(OH)-2’3,3’4’-5’-pentachlo biphenyls (1nM and 10nM), thalidomide (100nM and 0.01mM), bis(2-ethylhexyl)phthalate (0.001mM and 0.1mM), permethrin (100nM and 0.01mM), cyclopamine (100nM and 0.01mM), methoprene acid (0.001mM and 0.1mM)]; 1 Timepoint [48 hours].

ORGANISM(S): Mus musculus

SUBMITTER: Sone Hideko

PROVIDER: E-GEOD-37602 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The aim of this study is to identify microRNAs (miRNAs) transcriptionnally regulated by retinoic acid (RA). For that purpose we have used the RA-based treatment of the Acute Promyelocytic Leukemia (APL) as a model. This malignancy is characterised by a differentiation arrest of granulopoiesis at the promyelocytic stage. APL is molecularly associated with reciprocal translocations that always involve the retinoic acid receptor a (RARa). In the vast majority of APL cases, a t(15;17) chromosomal translocation fuses the genes encoding the promyelocytic leukemia protein PML and RARa. The resulting PML-RARa is a transcriptional repressor that impedes the expression of RA-regulated genes notably through an aberrant recruitment of transcriptional repressors and histone deacetylases. Consequently, these genes become insensitive to physiological doses (nanoM) of all-trans-retinoic acid (ATRA) but pharmacological doses (microM), overcome the PML-RARa-mediated repression and restore normal transcription and granulocytic differentiation. The restorative effects of RA can be reproduced in vitro in the NB4 cells, which were derived from an APL patient. These cells provide an excellent model to study the transcriptional deregulations that arise in APL and the molecular effects of the anti-cancerous RA-based treatment. We also used in this study the RA-resistant cells, namely NB4-LR1 and NB4-LR2 cells. The NB4-LR1 cells do transcriptionally respond to ATRA but do not maturate. In contrast, the NB4-LR2 cells show a clear defect in RA signaling, as they harbor a truncated form of PML-RAR protein that is not sensitive to pharmacological doses of RA. First, we plan to characterize miRNAs-repressed by PML-RAR. We reasoned that if some miRNAs are repressed by this protein, then pharmacological doses of RA should abolish this repression and lead to an increase in the level of expression of the corresponding miRNAs. NB4, NB4-LR1 and NB4-LR2 cells will thus be treated for 16h with ATRA (1 microM), and miRNAs profiles will be compared. We anticipate that, the expression of a potential miRNA-repressed by PML-RAR should be up-regulated by ATRA in both NB4 and NB4-LR1 cells but remain unchanged in NB4-LR2 cells. This expression pattern should in fact be similar to those observed for known RA-regulated genes, such as RARb, a well characterized target of the RARa and PML-RAR proteins. Importantly, this experimental procedure was already validated with the identification of a miRNA repressed by PML-RAR (patent Lecellier et al. #WO 2006/048553). MiRNAs candidates obtained will then be validated by chromatin immunoprecipitation using anti-RARa and anti-PML antibodies, followed by luciferase assays in presence or absence of ATRA. Keywords: retinoic acid-mediated gene regulation To characterize miRNAs regulated by PML-RAR.

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Identification of stage-specific gene expression signatures in response to retinoic acid during the neural differentiation of mouse embryonic stem cells

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