Temporal activation of XRCC1-mediated DNA repair is essential for muscle differentiation

Abstract

Transient DNA strand break formation has been identified as an effective means to enhance gene expression in living cells. In the muscle lineage, cell differentiation is contingent upon the induction of caspase-mediated DNA strand breaks, which act to establish the terminal gene expression program. This coordinated DNA nicking is rapidly resolved, suggesting that myoblasts may deploy DNA repair machinery to stabilize the genome and entrench the differentiated phenotype. Here, we identify the base excision repair pathway component XRCC1 as an indispensable mediator of muscle differentiation. Caspase-triggered XRCC1 repair foci form rapidly within differentiating myonuclei, and then dissipate as the maturation program proceeds. Skeletal myoblast deletion of Xrcc1 does not have an impact on cell growth, yet leads to perinatal lethality, with sustained DNA damage and impaired myofiber development. Together, these results demonstrate that XRCC1 manages a temporally responsive DNA repair process to advance the muscle differentiation program.

Keywords: DNA strand breaks; XRCC1; base excision repair; muscle differentiation.

Figure 1

DNA repair during early myoblast differentiation is associated with XRCC1 foci. (a) Co-staining for XRCC1 foci formation and in situ nick translation, to measure DNA polymerase activity, in C2C12 myoblast cells over differentiation time course. Scale bar, 10 μm. (b) Immunofluorescent staining for XRCC1 in caspase 3 inhibited (DEVD) differentiating C2C12 cells and counterstained using 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI). Scale bar, 10 μm. Images are representative from n=3 experimental replicates. (c) Data were quantified by counting the total number of foci per nucleus as represented in the histogram. (d) Targeted shRNA-mediated knockdown of CAD in differentiating C2C12 cells. The cells were induced to differentiate for 24 h and then were immunofluorescently stained for XRCC1. (e) Quantitative real-time PCR reveals an 80% reduction in CAD expression in the knockdown lines compared with the control. (f) Quantification of total XRCC1 foci per nucleus in proliferating cells (growth media) and after 24-h differentiation (24 h) is represented by histogram (n=3). Asterisks (***) indicate that the changes in foci formation between the two conditions indicated are statistically significant as determined by two-tailed Student's t-test analysis with P-value<0.01.

Figure 2

shRNA-mediated loss of Xrcc1 impedes myoblast differentiation. (a) Differentiation time course of shRNA-treated C2C12 myoblasts. Magnified inset panels included for 24-h differentiated conditions performed using the Photoshop CS3 software (Adobe Systems Inc., San Jose, CA, USA). (b) Real-time PCR demonstrates a reduction in Xrcc1 gene expression by 50% in shRNA-transfected cells at growth (n=3, one-tailed Student's t-test analysis with ***P-value<0.01 and *P<0.05). (c) Scoring the percentage of differentiated C2C12 cells (myosin heavy chain (MHC) cells/total nuclei) shows a decrease in differentiation after loss of XRCC1. Images are representative from n=3 experimental replicates. Scale bar, 50 μm.

Figure 3

Gene-targeted loss of XRCC1 leads to attenuation of myofiber development and perinatal lethality. (a) Wild-type (WT) and Myf5-Cre/ Xrcc1^flox/flox conditional knockout (cKO) of Xrcc1 pup images taken immediately post birth. Nuclear protein extraction from pooled hindleg muscles, and from liver as control, is used for western blot and probed for XRCC1 and GAPDH. Images representative from n=5 for each genotype. Western blot is representative from n=3 per genotype. (b) Longitudinal skeletal muscle sections stained for hematoxylin and eosin (H&E) or Masson’s Trichrome. Images representative from n=3. Scale bar, 200 μm. (c) Primary myoblasts from Xrcc1^flox/flox mice are treated with Cre-ad or Negative Ctl-ad and then induced to differentiate for 72 h. Images representative from n=5. Scale bar, 200 μm. Western blot probed for XRCC1 is from nuclear protein fraction isolated from Xrcc1^flox/flox primary myoblasts, either untreated, ultraviolet-treated to induce DNA damage or Cre-ad-treated to delete the Xrcc1 gene. Western blot is representative from n=3. Western blot for myosin heavy chain (MF20) in cytoplasmic protein lysates from Xrcc1^flox/flox primary myoblasts that were treated with either Cre-ad or Ctl-ad, and induced to differentiate for 72 h. Western blot is representative from n=3. (d) Western blot analysis from a time course treatment of Xrcc1^flox/flox primary myoblasts induced to differentiate by low-serum exposure. Blots representative from n=3.

Figure 4

Skeletal muscle gene expression profiles are altered with XRCC1 deletion. (a) Muscle-specific gene markers myoD, myogenin are unaffected by XRCC1 expression, whereas Mef2c and MCK inductions are significantly reduced in XRCC1-deleted cells (n=3, two-tailed Student's t-test analysis with ***P-value<0.01, **P-value<0.025, *P-value<0.05). (b) p21 gene expression profile during early differentiation. Xrcc1^flox/flox primary myoblasts infected with Ctl-ad (blue) or Cre-ad (red) and induced to differentiate up to 72 h displayed reduced p21 induction (c) ChIP-end point PCR of the p21 promoter region shows enhancement of XRCC1 binding during early differentiation of C2C12 cells compared with growth conditions (n=3). Caspase 3 inhibition (DEVD) leads to loss of XRCC1-p21 promoter binding. EF1alpha is used as a non-target genomic control for the ChIP experiment.

Figure 5

XRCC1 has a temporally sensitive requirement to mitigate DNA damage in differentiating myoblasts. (a) Knockout of Xrcc1 gene expression does not significantly affect myoblast proliferation potential. (b) Cell number counts quantified in graph. Images are representative from n=6. (c) Single-cell gel electrophoresis (Comet assay) was performed on differentiating Xrcc1^flox/flox primary myoblast cells treated with either Cre-ad or Ctl-ad. Following electrophoresis, cells were stained with SYBR green and visualized to assess the length of migration of DNA from the nucleus. Results show increased and persistent DNA damage in Xrcc1 knockdown cells, whereas control cells exhibit damage early in differentiation that is resolved over the differentiation time course. Images are representative from n=4 for each experimental condition; minimum number of cells counted =50 for each condition. Scale bar, 15 μm. (d) Cre-ad-mediated knockdown of Xrcc1 gene expression post induction of differentiation. Immunofluorescence staining for MHC was used to assess myotube formation. (e) The fusion Index graph quantifies myotube formation (n=3, two-tailed Student's t-test analysis with ***P-value<0.01). Scale bar, 20 μm. (f) Quantification of MHC protein at 3 h post induction of differentiation following adenovirus treatment. End point is 72 h post induction of differentiation (n=3, two-tailed Student's t-test analysis with *P-value<0.05). (g) Primary myoblasts isolated from MCK-Cre/Xrcc1^flox/flox transgenic mice show equivalent differentiation capacity to WT myoblasts. Scale bar, 200 μm. Graph shows that MHC expression is equivalent between WT and MCK-Cre/Xrcc1^flox/flox myoblasts differentiated for 72 h. (h) The XRCC1 protein expression profile is similar between WT and MCK-Cre/Xrcc1^flox/flox myoblasts differentiated for 72 h.