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Series GSE18125 Query DataSets for GSE18125
Status Public on Dec 22, 2011
Title Epigenetic regulation of Bmp2 and Smad6 in Ras-induced senescence
Organism Mus musculus
Experiment type Genome binding/occupancy profiling by high throughput sequencing
Expression profiling by array
Summary Epigenetically silenced Ink4a-Arf locus is activated by loss of H3K27me3 in cellular senescence, where secreted factor expression is also involved. Here we analyzed epigenome and transcriptome alteration during Ras-induced senescence using mouse embryonic fibroblast (MEF). Seventeen genes with H3K27me3 loss and H3K4me3 gain showed marked upregulation, including p16Ink4a and Bmp2, a secreted factor for BMP/SMAD signal. Smad6, specific BMP/SMAD pathway inhibitor, was identified as the only one gene showing de novo H3K27 trimethylation with H3K4me3, resulting in strong repression. Ras-activated cells senesced with SMAD1/5/8 phosphorylation, and they escaped from senescence with decreased SMAD1/5/8 phosphorylation when introducing Smad6 or knocking-down Bmp2.
Overall design Mouse embryonic fibroblasts were established from 13.5 embryonic day embryos of C57/B6. After cells were passaged twice (MEFp2), cells were infected with retroviruses for 48 hours. Then cells were exposed to 4 μg/mL peuromycin for selection during days 0-3, and were passed on days 3, 7, and 10.
Retroviral vectors for Ras was constructed by cloning cDNAs for wild type HRAS (RasG12) and mutated HRAS (RasV12) by reverse-transcription PCR products from HMEC and SK-BR3 cell RNA, respectively, with N-terminal FLAG tag into pMX vector that contains puromycin resistance gene. Mock pMX vector (mock), and vectors containing RasG12 and oncogenic RasV12 were transfected into plat-E packaging cells using FuGENE 6 Transfection Reagent (Roche, Germany) to prepare retroviruses. Smad6 cDNA with N-termainal 6x Myc tag was also cloned into pMX vector. To knock down Bmp2, double strand oligonucleotide DNA to express small hairpin RNA against Bmp2 (shBmp2) was cloned into RNAi-Ready pSIREN-RetroQ Vector (Clontech, CA). Viral packaging for Smad6 and shBmp2 retrovirus vectors was also done using plat-E cells.
For genome-wide transcription analysis, GeneChip Mouse Genome 430 2.0 Array (Affimetrix) was used. For global normalization, the average signal in an array was made equal to 100.
Chromatin immunoprecipitation (ChIP)-sequencing was performed. MEFp2 cells and cells with mock, RasG12 or RasV12 infection at day 10 were cross-linked with 1% formaldehyde for 10 min at room temperature and were prepared for ChIP. ChIP using anti-H3K4me3 (ab8580, abcam, rabbit polyclonal) or H3K27me3 (07-142, Upstate, rabbit polyclonal) antibody was performed as described previously. Sample preparation for ChIP-sequencing was performed according to the manufacturer's instructions (Ilumina), and sequencing was performed using Solexa Giga sequencer.
Contributor(s) Kaneda A, Aburatani H
Citation(s) 22072987
Submission date Sep 16, 2009
Last update date May 15, 2019
Contact name Atsushi Kaneda
Organization name The University of Tokyo
Department RCAST
Lab Genome Science Division
Street address 4-6-1 Komaba
City Meguro-ku
State/province Tokyo
ZIP/Postal code 153-8904
Country Japan
Platforms (2)
GPL1261 [Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array
GPL9185 Illumina Genome Analyzer (Mus musculus)
Samples (10)
GSM453339 MEFp2
GSM453340 Mock
GSM453341 RasG12
SRA SRP002312
BioProject PRJNA119455

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Supplementary file Size Download File type/resource
GSE18125_RAW.tar 371.5 Mb (http)(custom) TAR (of BED, CEL)
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Processed data included within Sample table
Raw data are available in SRA
Processed data provided as supplementary file

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