Caspase 3/caspase-activated DNase promote cell differentiation by inducing DNA strand breaks

Abstract

Caspase 3 is required for the differentiation of a wide variety of cell types, yet it remains unclear how this apoptotic protein could promote such a cell-fate decision. Caspase signals often result in the activation of the specific nuclease caspase-activated DNase (CAD), suggesting that cell differentiation may be dependent on a CAD-mediated modification in chromatin structure. In this study, we have investigated if caspase 3/CAD plays a role in initiating the DNA strand breaks that are known to occur during the terminal differentiation of skeletal muscle cells. Here, we show that inhibition of caspase 3 or reduction of CAD expression leads to a dramatic loss of strand-break formation and a block in the myogenic program. Caspase-dependent induction of differentiation results in CAD targeting of the p21 promoter, and loss of caspase 3 or CAD leads to a significant down-regulation in p21 expression. These results show that caspase 3/CAD promotes cell differentiation by directly modifying the DNA/nuclear microenvironment, which enhances the expression of critical regulatory genes.

Fig. 1.

Transient DNA strand breaks are detected early during C2C12 myoblast differentiation. (A) ISNT labels methanol-fixed C2C12 nuclei at 12 and 24 h after being switched from growth media (GM) to low-serum differentiation media (DM). Cells were stained for incorporated biotin-21-dUTP (green), myosin heavy chain (MHC; red), and DAPI (blue). (Error bars are SD.) (B) Phosphorylated H2AX foci are detected in differentiating C2C12 myoblasts at 12 and 24 h after low-serum–induced differentiation. Cells were fixed with 95% ethanol and 5% acetic acid and stained for P-H2AX (red) and DAPI (blue). (Error bars are SD.) (C) ISNT labels fixed primary myoblast nuclei at 12 h when switched to DM. Cells were stained for incorporated DIG-11-dUTP (red) and DAPI (blue).

Fig. 2.

DNA strand breaks are dependent on caspase 3 activity. (A) The caspase 3 inhibitor, z.DEVD.fmk, blocks the formation of DNA strand breaks detectable by ISNT. C2C12 myoblasts were induced to differentiate under low-serum conditions treated with either 10 μM z.DEVD.fmk or DMSO, and cells were fixed and stained as in Fig. 1A. (B) Formation of P-H2AX foci at 24 h of low-serum differentiation are diminished when myoblasts are treated with z.DEVD.fmk. Cells were treated as in A and fixed and stained as in Fig. 1B. (Error bars are SD; P < 0.001.) (C) Partial cleavage of ICAD corresponds to the transient formation of DNA strand breaks in differentiating C2C12 myoblasts. The ratio of p30 cleavage fragment to full-length ICAD increase by 4.6-fold at 12 h compared with growth. Protein lysates were collected from differentiating C2C12 myoblasts, and Western blot analysis for ICAD and CAD was performed. Tubulin was used as a loading control. (D) Caspase 3 inhibition impairs the partial cleavage of ICAD observed during differentiation. C2C12 myoblasts were differentiated for 12 h with 10 μM z.DEVD.fmk or DMSO. Protein lysates were collected, and Western blot analysis for ICAD was performed. (E) Expression of ICAD mutated at D117 impairs myoblast differentiation. Myoblasts were transfected with empty pMEV-2HA (control), wild-type–hICAD, or mutant D117E-hICAD. Stable expression was selected with G418, and then, myoblasts were induced to differentiate under low-serum conditions. Differentiation was assessed by the up-regulation of myosin heavy chain (red).

Fig. 3.

Stable knockdown of CAD in C2C12 myoblasts affects differentiation. (A) RT-PCR for CAD showing knockdown in stable C2C12 clones with shCAD compared with a C2C12 clone with a negative-control shRNA vector. RT-PCR for GAPDH was used as a loading control. (Error bars are SEM.) (B) Up-regulation of myosin heavy chain is impaired in the stable cell lines compared with negative control. Cells were induced to differentiate for 72 h, fixed, and stained for MHC (red). MHC-positive nuclei were counted from three independent experiments. (Error bars are SD.) (C) Myogenin expression during differentiation is not affected in the stable cell lines. Protein lysates were collected from differentiating stable cell lines, and Western blot analysis for myogenin expression was performed. Tubulin was used as a loading control. (D) Formation of phosphorylated H2AX foci at 24 h differentiation is diminished in shCAD stables compared with negative control. Cells were induced to differentiate for 24 h, fixed, and stained for P-H2AX.

Fig. 4.

Caspase 3/CAD inflicts DNA strand breaks within the promoter of the cell-cycle inhibitor p21. (A) Up-regulation of the cell-cycle inhibitor p21 is reduced in the stable shCAD cell lines compared with negative control. Cells were induced to differentiate for 24 h, fixed, and stained for p21 (red). Nuclei were counterstained with DAPI (blue). p21 positive nuclei were counted from three independent experiments. (Error bars are SD.) (B) p21 transcripts levels are reduced in the shCAD cell lines compared with negative control. RNA was isolated 24 h after the induction of differentiation and subject to semiquantitative RT-PCR. Multiplex PCR to p21 and GAPDH cDNA was run in triplicate for an n = 3 experiment. Percent reduction in p21 transcript in the CAD knock down (KD) myoblasts is compared with the negative control line. (C) CAD associates with the p21 promoter during C2C12 myoblast differentiation. ChIP using rabbit IgG (M) or an antibody against CAD (C) shows enrichment at the p21 promoter at 12 (2.5-fold) and 24 h (2.6-fold) after the induction of differentiation; no enrichment was observed of the myogenin promoter. (D and F) LM-PCR delineates the formation of DNA strand breaks within the p21 promoter of differentiating C2C12 and primary muscle cells. Genomic DNA was isolated from proliferating, differentiating, and apoptotic (staurosporine [STS]) muscle cells and subjected to LM-PCR. Primers specific to the p21 promoter allowed analysis of ∼200 bp before the transcriptional start site (TSS; n = 3). (D Inset and F Inset) Chemical inhibition of caspase 3 reduces the formation of strand breaks within the p21 promoter. Differentiating C2C12 and primary cells were treated with either 10 μM z.DEVD.fmk or DMSO; genomic DNA was extracted and subjected to LM-PCR (n = 3). Reduction in CAD expression impairs the formation of strand breaks within the p21 promoter in genomic DNA isolated from shCAD105 cells under low-serum conditions (D Inset; n = 2).(E and G) LM-PCR shows the absence of robust DNA strand breaks in the myogenin promoter during differentiation of C2C12 and primary muscle cells. Primers specific to the myogenin promoter allowed analysis of ∼200 bp before the TSS (n = 3).