DUX4c is up-regulated in FSHD. It induces the MYF5 protein and human myoblast proliferation.

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contractions of the D4Z4 repeat array in 4q35. We have previously identified a double homeobox gene (DUX4) within each D4Z4 unit that encodes a transcription factor expressed in FSHD but not control myoblasts. DUX4 and its target genes contribute to the global dysregulation of gene expression observed in FSHD. We have now characterized the homologous DUX4c gene mapped 42 kb centromeric of the D4Z4 repeat array. It encodes a 47-kDa protein with a double homeodomain identical to DUX4 but divergent in the carboxyl-terminal region. DUX4c was detected in primary myoblast extracts by Western blot with a specific antiserum, and was induced upon differentiation. The protein was increased about 2-fold in FSHD versus control myotubes but reached 2-10-fold induction in FSHD muscle biopsies. We have shown by Western blot and by a DNA-binding assay that DUX4c over-expression induced the MYF5 myogenic regulator and its DNA-binding activity. DUX4c might stabilize the MYF5 protein as we detected their interaction by co-immunoprecipitation. In keeping with the known role of Myf5 in myoblast accumulation during mouse muscle regeneration DUX4c over-expression activated proliferation of human primary myoblasts and inhibited their differentiation. Altogether, these results suggested that DUX4c could be involved in muscle regeneration and that changes in its expression could contribute to the FSHD pathology.

Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity.

BACKGROUND: it is now well established that hypoxia renders tumor cells resistant to radio- but also chemotherapy. However, few elements are currently available as for the mechanisms underlying this protection. RESULTS: in this study, physiological hypoxia was shown to inhibit apoptosis induced in HepG2 cells by etoposide. Indeed, hypoxia reduced DNA fragmentation, caspase activation and PARP cleavage. The DNA binding activity of 10 transcription factors was followed while the actual transcriptional activity was measured using specific reporter plasmids. Of note is the inhibition of the etoposide-induced activation of p53 under hypoxia. In parallel, data from low density DNA microarrays indicate that the expression of several pro- and anti-apoptotic genes was modified, among which are Bax and Bak whose expression profile paralleled p53 activity. Cluster analysis of data unravels several possible pathways involved in the hypoxia-induced protection against etoposide-induced apoptosis: one of them could be the inhibition of p53 activity under hypoxia since caspase 3 activity parallels Bax and Bak expression profile. Moreover, specific downregulation of HIF-1alpha by RNA interference significantly enhanced apoptosis under hypoxia possibly by preventing the hypoxia mediated decrease in Bak expression without altering Bax expression. CONCLUSION: these results are a clear demonstration that hypoxia has a direct protective effect on apoptotic cell death. Moreover, molecular profiling points to putative pathways responsible for tumor growth in challenging environmental conditions and cancer cell resistance to chemotherapeutic agents.

Gene expression and regulation in H2O2-induced premature senescence of human foreskin fibroblasts expressing or not telomerase.

We compared the DNA-binding activity of transcription factors and gene expression patterns in BJ human diploid fibroblasts (HDFs) expressing or not telomerase (hTERT) in stress-induced premature senescence (SIPS). Senescent BJ cells were also studied. Hydrogen peroxide (H2O2)-induced SIPS modulated gene expression in both BJ and hTERT-BJ1 cells. Increased p21(WAF-1) mRNA level was amongst the common gene expression changes in BJ and hTERT-BJ1 cells induced by SIPS. Telomerase expression markedly changed gene expression in non-stressful conditions. Expression patterns of senescent BJ cells partially overlapped those of BJ and hTERT-BJ1 cells in SIPS. The basal levels of DNA-binding activity of NF-kappaB and phosphorylated ATF-2 were different in BJ and hTERT-BJ1 cells. Both cell lines displayed a higher DNA-binding activity of p53 and HIF-1 72 h after H2O2 exposure. Our results indicate that similar mechanisms involving p21(WAF-1) and probably p53 are at work in BJ and hTERT-BJ1 HDFs under H2O2-induced SIPS, suggesting that generalized DNA damage rather than telomere length/telomerase plays a crucial role in H2O2induced SIPS. We propose that H2O2-induced SIPS involves a rearrangement of proliferative and apoptotic pathways. The marked changes in gene expression induced by telomerase suggest that apart from immortalization of HDFs, telomerase also alters the normal cellular functions but does not protect against SIPS.

No increase in senescence-associated beta-galactosidase activity in Werner syndrome fibroblasts after exposure to H2O2.

Normal human diploid fibroblasts (HDFs) exposed to a single H(2)O(2) subcytotoxic stress display features of premature senescence, termed stress-induced premature senescence (SIPS). In this work, our aim was to study SIPS in Werner syndrome (WS) fibroblasts, derived from a patient with WS, a disease resembling accelerated aging. The subcytotoxic dose for WS fibroblasts was found to be inferior to that of normal HDFs, indicating WS fibroblasts are more sensitive to hydrogen peroxide than normal HDFs. SA beta-gal activity has been shown to occur both in vitro and in vivo, and we studied the proportion of WS cells positive for SA beta-gal. Intriguingly, the percentage of positive cells did not increase with the dose of H(2)O(2) used. Contrary to other HDFs, the DNA-binding activity of p53 in WS fibroblasts did not increase in SIPS. We found, based on our results, that WS fibroblasts feature an altered stress response and do not reach SIPS from H(2)O(2). We suggest that the proportion of cells that in normal HDFs would enter SIPS instead die in WS fibroblasts. Last, we propose that aging derives from a loss of integrity of the chromatin structure, which occurs faster in WS patients.