{"owner": "ArrayExpress Uploader", "pop_total": 0, "id": 5828, "factors": [{"GSM453339": {"ANTIBODY": "not specified", "TREATMENT": "not specified"}}, {"GSM453340": {"ANTIBODY": "not specified", "TREATMENT": "infected with mock pMXs virus"}}, {"GSM45334": {"ANTIBODY": "not specified", "TREATMENT": "infected with wild type RasG12-expressing retrovirus"}}, {"GSM453342": {"ANTIBODY": "not specified", "TREATMENT": "infected with activated RasV12-expressing retrovirus"}}], "ownerprofile_id": "arrayexpress_sid", "platform": 6, "summary_wrapped": "Epigenetically silenced Ink4a-Arf locus is activated by loss of H3K27me3 in cellular senescence, where secreted factor expression is also...", "pubmed_id": 22072987, "geo_gse_id": "E-GEOD-18125", "owner_profile": "/profile/8773/arrayexpressuploader", "factor_count": 2, "sample_count": 4, "tags": ["cell", "chromatin", "fibroblast", "genome", "plat"], "lastmodified": "Dec.12, 2014", "is_default": false, "geo_gds_id": "", "slug": "epigenetic-regulation-of-bmp2-and-smad6-in-ras-ind", "geo_id_plat": "E-GEOD-18125_A-AFFY-45", "name": "Epigenetic regulation of Bmp2 and Smad6 in Ras-induced senescence", "created": "Nov.11, 2014", "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. 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 \u03bcg/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.", "source": "http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-18125", "species": "mouse", "sample_source": "http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-18125/samples/"}