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. 2011 Jul 15;10(14):2355-63.
doi: 10.4161/cc.10.14.15948. Epub 2011 Jul 15.

Coordination of cell cycle, DNA repair and muscle gene expression in myoblasts exposed to genotoxic stress

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Coordination of cell cycle, DNA repair and muscle gene expression in myoblasts exposed to genotoxic stress

Marta Simonatto et al. Cell Cycle. .

Abstract

Upon exposure to genotoxic stress, skeletal muscle progenitors coordinate DNA repair and the activation of the differentiation program through the DNA damage-activated differentiation checkpoint, which holds the transcription of differentiation genes while the DNA is repaired. A conceptual hurdle intrinsic to this process relates to the coordination of DNA repair and muscle-specific gene transcription within specific cell cycle boundaries (cell cycle checkpoints) activated by different types of genotoxins. Here, we show that, in proliferating myoblasts, the inhibition of muscle gene transcription occurs by either a G 1- or G 2-specific differentiation checkpoint. In response to genotoxins that induce G 1 arrest, MyoD binds target genes but is functionally inactivated by a c-Abl-dependent phosphorylation. In contrast, DNA damage-activated G 2 checkpoint relies on the inability of MyoD to bind the chromatin at the G 2 phase of the cell cycle. These results indicate an intimate relationship between DNA damage-activated cell cycle checkpoints and the control of tissue-specific gene expression to allow DNA repair in myoblasts prior to the activation of the differentiation program.

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Figures

Figure 1
Figure 1
Diverse genotoxic agents induce cell cycle arrest at different boundaries and display different kinetics of DNA repair. C2C7 mouse myoblasts were exposed to Methylmethanesulfonate (MMS) 75 µM, Doxorubicin (Dox) 0.4 µM and Etoposide (Eto) 0.5 µM before incubation in differentiation medium (DM) for 24 h. See scheme on top. (A) The cell cycle profle was analyzed by cytofuorimetric analysis in the conditions described above or in control (untreated) cells cultured in GM or shifted in DM for 18 h. (B) The kinetic of repair of the DNA lesions caused by the different genotoxins was monitored by comet assay at different time points (T) after shifting the cells in DM. Representative fields of no-treated cells (NT) MMS-, Eto- and Dox-treated cells at different time points. (C) Quantification of the repair in the same experiment shown in (B) by plotting the average of the tail moment (calculated using Tritek CometScore™) of at least 100 cells per experimental point. This experiment is representative of multiple, independent experiments, which consistently show the same pattern of repair for each genotoxic agent, despite of the variability of the values that is intrinsic to each comet assay. (D) Human myoblasts were treated as described above and the expression levels of myogenin at different time points after DNA damage were assessed by real time PCR.
Figure 2
Figure 2
Transcription profle of myoblasts exposed to Doxorubicin treatment. Primary Human Skeletal Muscle Myoblasts (HSMBs) were exposed for 18 h to Doxorubicin 0.4 µM while growing in GM and then shifted in DM for additional 24 h. Total RNA was extracted with Trizol® as described in the data sheet (Invitrogen). A microarray was performed using Sentrix Human Ref-8 BeadChip (Illumina). In the panel is shown the analysis of the differentially expressed transcripts divided in upregulated (left) and downregulated (right). Bar charts represent the functional categories statistically more significant (≥ 2-fold change) in our groups of genes that include GO Biological Process, GeneGo Process Network and GeneGo Pathway Maps. Analyzed using MetaCore™ software from GeneGo Inc.
Figure 3
Figure 3
Different mechanisms are responsible for the differentiation checkpoint induced by genotoxic agents that activate the G1 or G2 checkpoints. C2C12 cells were untreated or treated with Doxorubicin and MMS, as shown in Figure 1. When indicated, 5 mM caffeine was added 30 min before drug exposure. After genotoxic treatments cells, were shifted to differentiation medium for an additional 24 h. (A) ChIP analysis was performed to monitor MyoD binding and the H3K9/14 acetylation status (AcH3) to the chromatin of myogenin promoter or muscle creatin kinase (MCK) enhancer after MMS treatment. (B) ChIP analysis was performed as in (A) after Dox treatment. (C) The effect of caffeine treatment on the cell cycle was monitored by cytofluorimetric analysis. (D) The phosphorylation of p53 on Serine15 was assessed by protein gel blot on cells collected after genotoxins exposure. Tubulin was used as a loading control. (E) Expression levels of muscle-specific proteins (Myogenin and Myosin Heavy Chain-MyHC) were monitored by protein gel blot. Tubulin was used as a loading control.
Figure 4
Figure 4
IGF-1 bypass the G1-differentiation checkpoint imposed by MMS treatment. Human Skeletal Muscle Myoblasts (HSMBs) were exposed to Dox and MMS in the presence or in the absence of 5 mM caffeine and 1ug/ml IGF-1 then shifted to DM. Cells were immunostained for MyHC and DAPI (A).The expression of myogenin was assessed by protein gel blot. Tubulin was used as a loading control (B). (C) C2C12 cells were treated as in (B), and ChIP analysis was performed to monitor the H3 tri-methylation in lysine 4 (MetK4) to the chromatin of muscle creatin kinase (MCK) enhancer.

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References

    1. Harper JW, Elledge SJ. The DNA damage response: ten years after. Mol Cell. 2007;28:739–745. doi: 10.1016/j.molcel.2007.11.015. - DOI - PubMed
    1. Hartwell LH, Kastan MB. Cell cycle control and cancer. Science. 1994;266:1821–1828. doi: 10.1126/science.7997877. - DOI - PubMed
    1. Kastan MB, Bartek J. Cell cycle checkpoints and cancer. Nature. 2004;432:316–323. doi: 10.1038/nature03097. - DOI - PubMed
    1. Bartek J, Lukas C, Lukas J. Checking on DNA damage in S phase. Nat Rev Mol Cell Biol. 2004;5:792–804. doi: 10.1038/nrm1493. - DOI - PubMed
    1. Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009;461:1071–1078. doi: 10.1038/nature08467. - DOI - PMC - PubMed

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