p53 transcriptionally represses myogenin. (a) Schematics of the control- and p53-ER fusion constructs. Protein translation levels are modulated by synthetic upstream open reading frames as shown by black ovals (details in ). Arrow: transcription start site (TSS); mKate: red fluorescent protein; 2A: ribosomal slippage sites; BFP: blue fluorescent protein. (b) Transcriptional activity of p53-ER fusion constructs was determined by measuring expression of p53-activated target genes. Nuclear localization of the ER fusion proteins was induced by 4-hydroxytamoxifen (4OHT). Ethanol treatment served as the uninduced control. Quantitative PCR (Q-PCR) graph represents transcription level relative to that of the control in the absence of 4OHT induction. The fusion proteins were expressed from high to low levels as represented by the black gradient bars here and in all following figures. (c) Q-PCR analysis shows dose-dependent repression of myogenin in response to ectopic p53 in RD cells. (d) Immunoblotting (IB) shows corresponding reduction of myogenin protein upon p53 activation. (e) Schematic of p53 mutants, p53, p53, and p53. TAD: transcriptional activation domain; PRD: proline-rich domain; DNA binding: DNA-binding domain; Tet: tetramerization domain; Basic: basic domain. (f) Q-PCR analyses show p21 activation and myogenin repression in response to ectopic p53 and its mutants at 48 h of 4OHT induction. Ectopic expression of mouse p21 served as a negative control for myogenin mRNA level in RD cells. Quadruple mutation (QM): p53. (g) IBs show p21 activation and myogenin repression in response to ectopic p53 and its mutants at 48 h of 4OHT induction. Ectopic p21 in RD cells led to accumulation of myogenin at the protein level and served as a positive control for IB. QM: p53

The reduction of myogenin protein in response to p53 activation is independent of cell cycle phases. (a) Distribution of cell cycle phases was quantified in RD cells ectopically expressing control, p53, or p21. The flow cytometry plot shows G1, S, and G2 phases based on BrdU and PI staining. (b) Reduction of myogenin protein in each cell cycle phase was evaluated by co-immunostaining of myogenin and BrdU, followed by flow cytometry analysis. Quadrants were drawn to denote a MyoG-H population in either G1 or S phase at 48 h of 4OHT induction. Ectopic expression of p21 served as a positive control. FSC: forward scatter. (c) Summary of the percentage of cells with MyoG-H expression in either G1 or S phase in response to ectopic p53 or p21 expression. (d) Immunoblotting (IB) shows that p53 activation induced expression of endogenous p21 and repressed myogenin expression in contrast to ectopic expression of p21, which led to accumulation of myogenin at 24 h of 4OHT induction. (e) Expression of myogenin protein in response to ectopic p53 and its mutants is summarized as the percentage of MyoG-H cells in either G1 or S phase at 48 h of 4OHT induction

p53 binds to the myogenin promoter and regulates its transcription in response to genotoxic stress. (a) Schematics of putative p53 or p63 response elements (REs) on the mouse and human myogenin promoter with p53 and p63 ChIP-seq profiles,^, respectively. Arrow: transcription start site (TSS); Myog or MYOG: myogenin gene; bg: background signal. (b) Protein or RNA expression kinetics of p53 and myogenin from 2 to 96 h post IR in C2C12 myoblasts maintained under either growth or differentiation condition. (c) ChIP analysis of p53 binding to mouse myogenin 2560 site at 6 and 48 h post IR in C2C12 myoblasts maintained under either growth or differentiation condition. ChIP background was measured by binding of the p53 antibody to a gene desert region. p53 enrichment on the p21 promoter, p21, served as a positive control. ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. (d) ChIP analysis of p53 binding to the human myogenin promoter. ChIP was performed with an anti-ER antibody of RD cells ectopically expressing ER fusion of the control, p53, or p53^R245W. p53^R245W is the mouse equivalent of human p53^R248W expressed in RD cells and is defective in DNA binding. Fold enrichment relative to the control is shown as bold numbers on the top of each bracket. ChIP background was measured by binding of the ER-fusion proteins to a gene desert region. p53 enrichment on the p21 promoter, p21, served as a positive control

Repression of myogenin by p53 is partially mediated through a distal enhancer region upstream of the mouse myogenin gene. (a) Schematics of the distal enhancer region upstream of the mouse myogenin gene. Black squares: R1, R2, and R3 enhancer elements; arrow: transcription start site (TSS). The p53 consensus binding sequence is characterized as two half sites of RRRCWWGYYY (R=purine, W=adenine or thymine, and Y=pyrimidine) with a spacer of 0–13 base pairs between each half site. Mouse myogenin p53 RE at−2560 site is shown in the bracket and mutations at the consensus sites are underlined as 2970–2328 A/T or G/C. (b) p53-ER transcriptional activity at 24 h of 4OHT induction was validated by luciferase reporter assays in RD or 293 cells. The reporter 2970-TSS includes the fragment from −2970 to TSS. The reporter 2970-2328 includes the fragment from −2970 to −2328. pGL3 vector and 4RTK-Luc served as negative controls. p21-Luc served as a positive control. (c) ChIP analysis of histone H3K27ac enrichment to mouse myogenin enhancer elements at 48 h post IR in C2C12 myoblasts maintained under either growth or differentiation condition. ChIP background was measured by binding of the H3K27ac antibody to a gene desert region. *P<0.05; ***P<0.001. (d) ChIP analysis of polymerase II enrichment to mouse myogenin R2 (MyoG-2560) and TSS at 48 h post IR in C2C12 myoblasts maintained under either growth or differentiation condition. ChIP background was measured by binding of the polymerase II antibody to a gene desert region. **P<0.01; ****P<0.0001

Repression of myogenin by p53 leads to reduction of late but not early differentiation markers. (a) In response to ectopic p53, expression of an early differentiation marker, desmin, was quantitatively analyzed by co-immunostaining myogenin in RD cells and followed by flow cytometry analysis at 48 h of 4OHT induction. Two gates were drawn as myogenin-low and desmin-high (MyoG-L Des-H) on the left and myogenin-high and desmin-high (MyoG-H Des-H) on the right. (b) Summary of the percentage of RD cells in either MyoG-H Des-H or MyoG-L Des-H. QM: p53. (c) Q-PCR analysis of desmin expression from 2 to 96 h post IR in C2C12 myoblasts maintained under differentiation condition. (d) Expression of MyHC in RD cells in response to ectopic p53 and mutants was analyzed by immunofluorescence (IF) and quantified by the Differentiation Index, which is the percentage of MyHC-positive cells above the total number of nuclei. Ectopic p21 served as a positive control. (e) Immunoblotting shows expression of myogenin and MyHC from 6 to 96 h post IR in C2C12 myoblasts maintained under differentiation condition

p53-mediated repression of myogenin reduces post-mitotic nuclear abnormality in terminally differentiated cells in response to acute DNA damage. (a) Experimental design and summary of results on p53-null MEFs for assays of myogenin function in either a p53-dependent or p53-independent manner. IR: ionizing radiation. Red-cross sign: p53 is unable to repress ectopic myogenin. Endogenous expression is shown as hashed grey and ectopic expression as filled grey. Myogenic differentiation was quantified using both Differentiation Index (the percentage of MyHC⁺ cells above the total number of nuclei) and Fusion Index (the percentage of the number of nuclei in MyHC⁺ cells containing at least three nuclei above the total number of nuclei). The percentage of abnormal nuclei in differentiated muscle cells (MyHC⁺) is summarized as either high or low in abnormal nuclei. (b) Examples of nuclear abnormality observed in irradiated cells versus non-irradiated cells. Using immunofluorescence (IF), nuclei were scored as abnormal if they were micronuclei, macronuclei, lobulated nuclei, or fragmented nuclei. Scale bar: 20 μm. (c) Myogenic differentiation induced by MyoD, myogenin, or both in the presence of ectopic p53 or the QM mutant, p53. (d) The percentage of abnormal nuclei in MyHC⁺ cells expressing a high or medium level of MyoD or myogenin in the presence of ectopic p53 or the QM mutant. Statistical significance of reduction in numbers of abnormal nuclei relative to that of the control was determined by a two-tailed Student's t-test. (e) The percentage of abnormal nuclei in MyHC⁺ cells induced by MyoD, myogenin, or both in the presence of ectopic p53 or the QM mutant. (f) Live-cell images show cells undergoing myogenic conversion and acquiring post-mitotic nuclear defects. Images are shown in three channels from a single frame at 36 h post IR. DHB-mVenus: a fragment of DHB fused to a yellow fluorescent protein. H2B-mTurquoise: a histone H2B fused to a cyan fluorescent protein. MyoD-mCherry: MyoD fused to a red fluorescent protein. Solid white block arrows indicate a cell in G1 and open white block arrows indicate a cell in G2. White slender arrows indicate two MyoD-positive cells showing severely fragmented nuclei arrested in G1 phase at 36 h post IR after one or two rounds of mitosis

p53-mediated repression of myogenin regulates muscle differentiation in response to genotoxic stress. Muscle differentiation is represented in black lines and p53-mediated regulation in heavy red lines. Skeletal muscle differentiation is orchestrated by members of the MyoD family of transcription factors. MyoD determines the myogenic lineage and acts upstream of p21 and myogenin.^{, , ,} Under normal conditions, expression of both p21 and myogenin is critical to establish the state of irreversible cell cycle arrest and terminal differentiation.^{, ,} In contrast to the cell cycle arrest that promotes terminal differentiation under non-stressed conditions, it is known that cell cycle arrest induced by DNA damage is associated with decreased myogenic differentiation. In addition to the previously characterized mechanism that genotoxic stress inactivates MyoD function through a p53-independent mechanism,^{, , ,} we describe a direct regulatory mechanism by which p53 represses myogenin in response to genotoxic stress. This repression of myogenin leads to reduction of late-stage differentiation and prevents formation of post-mitotic nuclear abnormality in terminally differentiated muscle cells. We propose that the regulation of myogenin plays a critical role not only in driving terminal differentiation but also in maintaining cellular integrity in terminally differentiated muscle cells by virtue of its conditional repression by p53.