The myogenic factor MyoD regulates a complex program of gene expression involving a high number of muscle specific genes and cell cycle regulators. The MyoD-dependent induction of the cell cycle inhibitor p57 presents a complex and multilevel regulation. We previously found that MyoD causes the release of a repressive chromatin loop, mediated by CTCF, between the promoter of p57 and a 150 kb-distant regulatory element, KvDMR1.
Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of various proteins and participates in the regulation of chromatin structure during DNA repair. PARP1 also regulates transcription through complex mechanisms not completely understood. It has been reported that CTCF physically interacts with and is a target of the poly(ADP-ribose)polymerase PARP1, whose activity is required for CTCF-mediated long-range chromatin insulation. Moreover, increasing evidence is accumulating that PARP1 and PARylation modulate the activity of several chromatin modifying enzymes. We have recently observed that PARP1 is required for keeping p57 under control, that PARP1 binds to KvDMR1 and that MyoD phsically interacts with PARP1. Moreover, preliminary results suggest that PARP1 also affects some muscle-specific genes.
In the present project I plan to investigate the role of PARP1 activity in p57 regulation, through the following tasks:
- Analyse the chromatin accumulation of Poly(ADP)ribose (PAR) and histone modifications on the p57 regulatory regions during differentiation in the presence and in the absence of PARP1.
- Determine the PARylation status of CTCF during differentiation
- Analyze the effects of PARP inhibitors on p57 expression and on the three-dimensional structure of the locus.
- Determine the role of MyoD in PARP1 activation
This study will have a scientific impact not only in the field of the epigenetic mechanisms governing muscle differentiation, but also in the biology of transcriptional regulation by PARP1.
The study of p57 regulation in muscle cells not only represents a valuable model of the multiple and complex strategies devised by mammals for fine-tuning gene transcription during growth and differentiation, but also will have both diagnostic and therapeutic potential in the next future. The downregulation of p57 expression has been linked to cancer progression and to overgrowth diseases such as Beckwith-Wiedemann syndrome (BWS), as well as to focal hyperinsulinism and preeclampsia syndrome (1). Conversely, excess of p57 expression characterizes growth restriction syndromes such as Silver Russel (SRS), intra uterine growth restriction (IUGR) and IMAGe syndromes, and is associated with type 2-diabetes risk (1). The occurrence of mutations or epigenetic changes at critical regulatory sequences, as well as the possible alterations of trans-acting factors interacting with these regions, could be used for the diagnosis of diseases associated with abnormal p57 expression. Moreover, unravelling the functions of epigenetic factors and of their mutual interplay promises the possibility of employing epigenetic drugs in order to restore the correct pattern of p57 expression.
In addition to this, a more complete understanding of the multiple molecular mechanisms by which MyoD governs myogenesis, and of their interplay, will give an important contribution to expand our basic knowledge of the transition from proliferation to differentiation, a critical step in muscle development. The identification of new co-regulators of MyoD function is also important in light of the obvious interest in muscle regeneration, a process that involves the activation and differentiation of muscle precursors and that is the object of intense studies aimed at manipulating the differentiation process in order to devise new treatments of myopathies. Moreover, it is worth mentioning that some muscle pathologies, such as rhabdomyosarcoma, involve the defective expression of several MyoD targets (2). This has been associated to the aberrant functioning of several molecular pathways, as well as to some genetic and epigenetic alterations. Since MyoD is normally expressed and shows a widespread binding to chromatin in this tumour, a better understanding of the interplay between MyoD and chromatin modifiers will be helpful in clarifying the causes of the impaired MyoD activity in rhabdomyosarcomas.
Finally, it is worth mentioning that inhibition of PARP activity provides therapeutic benefits in cancer and inflammation-related pathologies and that some pharmacological PARP inhibitors are being used in clinical trials for the therapy of specific cancers (3). Remarkably, PARP inhibitors have been also shown to reduce muscle damage after ischemia/reperfusion (4), implying their possible use as therapeutic agents after muscle injury. Therefore, the attainment of an integrated picture of the complex and multiple roles of PARP-activity in muscle, as well as in other tissues, is extremely important for increasing our awareness of the pleiotropic effects of modulating PARP function.
1. Rossi, M. N., Andresini, O., Matteini, F., and Maione, R. (2018) Transcriptional regulation of p57(kip2) expression during development, differentiation and disease. Frontiers in bioscience 23, 83-108
2. Keller, C., and Guttridge, D. C. (2013) Mechanisms of impaired differentiation in rhabdomyosarcoma. The FEBS journal 280, 4323-4334
3. Rouleau, M., Patel, A., Hendzel, M. J., Kaufmann, S. H., and Poirier, G. G. (2010) PARP inhibition: PARP1 and beyond. Nature reviews. Cancer 10, 293-301
4. Crawford, R. S., Albadawi, H., Atkins, M. D., Jones, J. E., Yoo, H. J., Conrad, M. F., Austen, W. G., Jr., and Watkins, M. T. (2010) Postischemic poly (ADP-ribose) polymerase (PARP) inhibition reduces ischemia reperfusion injury in a hind-limb ischemia model. Surgery 148, 110-118