Poly(ADP-ribose)polymerase-1 (PARP-1) is a predominantly nuclear enzyme that exerts numerous functions in cellular physiology and pathology, from maintenance of DNA stability to transcriptional regulation. Through a proteomic analysis of PARP-1 co-immunoprecipitation complexes, we identified Mitofilin, a mitochondrial protein, as a new PARP-1 interactor. This result prompted us to further investigate the presence and the role of the enzyme in mitochondria. Using laser confocal microscopy and Western blot analysis of purified mitochondria, we demonstrated the mitochondrial localization of a fraction of PARP-1. Further, the effects of overexpressing or down-regulating Mitofilin showed that this protein promotes and is required for PARP-1 mitochondrial localization. We also report several lines of evidence suggesting that intramitochondrial PARP-1 plays a role in mitochondrial DNA (mtDNA) damage signaling and/or repair. First, we show that PARP-1 binds to different regions throughout the mtDNA. Moreover, we demonstrated that the depletion of either PARP-1 or Mitofilin, which abrogates the mitochondrial localization of the enzyme, leads to the accumulation of mtDNA damage. Finally, we show that DNA ligase III, known to be required for mtDNA repair, participates in a PARP-1-containing complex bound to mtDNA. This work highlights a new environment for PARP-1, opening the possibility that at least some of the nuclear functions of the enzyme can be also extended to mtDNA metabolism.

As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way.To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65tpr-met) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65tpr-met highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand–receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65tpr-met kinase. These data indicate that a met kinase signal released from differentiation-dependent control provides a negative stimulus for the onset of myogenic differentiation.

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The bHLH transcription factor MyoD, the prototypical master regulator of differentiation, directs a complex program of gene expression during skeletal myogenesis. The up-regulation of the cdk inhibitor p57kip2 plays a critical role in coordinating differentiation and growth arrest during muscle development, as well as in other tissues. p57kip2 displays a highly specific expression pattern and is subject to a complex epigenetic control driving the imprinting of the paternal allele. However, the regulatory mechanisms governing its expression during development are still poorly understood. We have identified an unexpected mechanism by which MyoD regulates p57kip2 transcription in differentiating muscle cells. We show that the induction of p57kip2 requires MyoD binding to a long-distance element located within the imprinting control region KvDMR1 and the consequent release of a chromatin loop involving p57kip2 promoter. We also show that differentiation-dependent regulation of p57kip2, while involving a region implicated in the imprinting process, is distinct and hierarchically subordinated to the imprinting control. These findings highlight a novel mechanism, involving the modification of higher order chromatin structures, by which MyoD regulates gene expression. Our results also suggest that chromatin folding mediated by KvDMR1 could account for the highly restricted expression of p57kip2 during development and, possibly, for its aberrant silencing in some pathologies.

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