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Figures
- Fig. 1.
The p53 pathway is not relevant to DUX4 activity in vitro. (A) Viability of iC2C12-DUX4 cell lines, which express DUX4 in response to different doses of doxycycline and other factors constitutively from the murine stem cell virus (MSCV) long terminal repeat (LTR), or empty vector (EV). Viability in the presence of doxycycline (Dox) is normalized to that in its absence. n=8 biological replicates (separate wells) per group. Data presented as means±s.e.m. t-test: ****P<0.0001. (B) Western blots for DUX4 (top), p53 (middle) and GAPDH (bottom). Representative western blot image from 1 of 4 biological replicates is shown. (C) RTqPCR for Cdkn1a expression under various conditions, normalized to Gapdh expression. Data presented as means±s.e.m., n=3, t-test: ****P<0.0001.
- Fig. 2.
Lack of transcriptional response to DUX4 in p53 target genes. (A) Heatmap of log2 expression values for microarray data for DUX4 targets in mouse C2C12 cells upon doxycycline (DOX)-induced DUX4 expression. Each replicate is shown independently and the amount of time between induction and harvest is indicated above each column. DUX4 targets (upper panels) and Cdkn1a, which is both a DUX4 target and a p53 target in mouse cells (middle panel), are reproducibly upregulated, whereas the remaining traditional p53 targets show no discernable pattern of regulation (lower panels). (B) Heatmap of log2 expression values for RNA-seq data for DUX4 targets, grouped as in A. (C) Microarray experiments in human myoblasts cells. Gene lists are grouped as in A and B, with the exception of Cdkn1a, which is grouped with the p53 targets as it is not a DUX4 target in human cells. (D) RNA-seq experiments in the human LHCNM2 cell line. (E) RNA-seq experiments in the human MB135 cell line. Note that, in C, D and E, p53 targets are not induced (lower panels) despite the strong induction of DUX4 targets (upper panels). See the reference list for references stated in the figures.
- Fig. 3.
p53 status does not impact survival of iDUX4[2.7] animals to birth. Summary of genotypes observed from a backcross of p53-knockout males to p53 heterozygous females carrying the iDUX4[2.7] transgene. Expected values are based on total numbers, assuming no loss of viability. Note that no male carriers were observed, neither in the heterozygous nor homozygous p53-null state. Testing the hypothesis that p53 affects survival, using the 4 classes of male progeny, P=0.74 (Fisher's exact test); therefore, the null hypothesis (p53 does not affect survival of iDUX4[2.7] mice) is assumed. ‘D’=presence of iDUX4[2.7] transgene, versus Y=Y-chromosome or ‘+’=wild-type X-chromosome; p=p53-null versus ‘+’=wild-type p53 allele, i.e. p/p=null for p53.
- Fig. 4.
Muscle progenitors are killed by DUX4 on the p53 mutant background. (A) Photomicrographs of homozygous fibro/adipogenic (PDGFRα+) and myogenic (Itga7+) progenitors from male 3-week-old p53-knockout mice; (top) iDUX4[2.7] mice under control expansion conditions or (bottom) iDUX4[2.7] mice exposed to doxycycline (Dox) to induce DUX4 expression. Scale bars: 100 µm. (B) Viability assay for the same cells, exposed to different doses of doxycycline; n=8. Luminescence detects ATP, measuring viability of the cultures. Doxycycline induces loss of viability in both myoblasts and fibro/adipogenic progenitors. Data presented as means±s.e.m. (P<0.0001 by two way ANOVA/Sidak post hoc for both studies).
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