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| 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
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| 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.
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| 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.
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| Contributor(s) |
Kaneda A, Aburatani H |
| Citation(s) |
22072987 |
| Submission date |
Sep 16, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Atsushi Kaneda |
| E-mail(s) |
kaneda@genome.rcast.u-tokyo.ac.jp
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| Organization name |
The University of Tokyo
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| Department |
RCAST
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| Lab |
Genome Science Division
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| Street address |
4-6-1 Komaba
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| City |
Meguro-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
153-8904 |
| Country |
Japan |
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| Platforms (2) |
| GPL1261 |
[Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array |
| GPL9185 |
Illumina Genome Analyzer (Mus musculus) |
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| Samples (10)
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| Relations |
| SRA |
SRP002312 |
| BioProject |
PRJNA119455 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE18125_RAW.tar |
371.5 Mb |
(http)(custom) |
TAR (of BED, CEL) |
SRA Run Selector |
| Processed data included within Sample table |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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