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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 11, 2015 |
| Title |
RNA-seq analysis of acute Rb1 deletion in adult mouse tissues |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Abstract:The pRb tumor suppressor protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb-loss are largely unexplored. We have acutely ablated Rb in adult mice and conducted quantitative analysis of RNA and proteomic changes in colon and lung, where Rb-loss was sufficient or insufficient to induce ectopic proliferation respectively. As expected, RbKO caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but unexpectedly their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb-loss are coupled with proliferation, but uncoupled from transcription. The proteomic changes in common between RbKO tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and OXPHOS function. RBKO cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb-loss altered mitochondrial pyruvate oxidation from 13C-Glucose through the TCA cycle in both cells and mouse tissues. Consequently, RBKO cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.
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| Overall design |
Design: Two tissues (lung and colon) from adult control (Rb1 wild type) or variable (Rb1 acute knockout). We examined the consequences of pRb loss in an in vivo model. Mice were bred to introduce a tamoxifen inducible form of Cre recombinase (ROSA26Sortm1(cre/Esr1)Tyj ) into the genetic backround of either Rb wild type (Rb+/+) or Rb floxed (Rbf/f) alleles. These animals were then injected with tamoxifen, ablating Rb in Rbf/f mice and generating RbKO tissues. Lung and colon tissues were harvested 96 hrs after the tamoxifen injections and analyzed. Each cohort used 3 biological replicates.
Methods: RNA extraction: Ninety six hours following the last injection of tamoxifen mice were processed were sacrificed and dissected tissues flash frozen in liquid nitrogen for RNA extraction. RNA was extracted using the TRIzol-choloroform method and then DNase treated. RNA quality was validated using a bioanalyzer. Library construction and RNA-squencing was performed at the MGH NextGeneration Sequencing facility. Three mice were used per genotype, taking matched tissues from each mouse.
Methods: Alignment: Sequenced libraries were mapped to the mouse genome (mm10) using TOPHAT (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks, Trapnell C et al, Nature protocols 2012, 7, 562-578, doi:10.1038/nprot.2012.016). A RefSeq GTF file was used to annotate the mapped transcripts. FPKM matrixes and differentially regulated gene lists were generated using the Cufflinks-Cuffmerge-Cuffdiff pipeline (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks, Trapnell C et al, Nature protocols 2012, 7, 562-578, doi:10.1038/nprot.2012.016).
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| Contributor(s) |
Nicolay BN, Morris R, Dyson NJ, Kottakis F |
| Citation(s) |
26314710 |
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| Submission date |
Aug 10, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Robert Morris |
| E-mail(s) |
morris.robert@mgh.harvard.edu
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| Phone |
617-724-6552
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| Organization name |
Mass. General Hospital Cancer Center
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| Street address |
149 13th Street
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| City |
Charlestown |
| State/province |
Massachusetts |
| ZIP/Postal code |
02129 |
| Country |
USA |
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| Platforms (1) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA292461 |
| SRA |
SRP062249 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE71924_All_samples_FPKM.xlsx |
2.8 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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