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1.  MyoD regulates p57kip2 expression by interacting with a distant cis-element and modifying a higher order chromatin structure 
Nucleic Acids Research  2012;40(17):8266-8275.
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.
doi:10.1093/nar/gks619
PMCID: PMC3458561  PMID: 22740650
2.  Polyomavirus Large T Antigen Induces Alterations in Cytoplasmic Signalling Pathways Involving Shc Activation 
Journal of Virology  1999;73(2):1427-1437.
It has been extensively demonstrated that growth factors play a key role in the regulation of proliferation. Several lines of evidence support the hypothesis that for the induction of cell cycle progression in the absence of exogenous growth factors, oncogenes must either induce autocrine growth factor secretion or, alternatively, activate their receptors or their receptor substrates. Cells expressing polyomavirus large T antigen (PyLT) display reduced growth factor requirements, but the mechanisms underlying this phenomenon have yet to be explored. We conducted tests to see whether the reduction in growth factor requirements induced by PyLT was related to alterations of growth factor-dependent signals. To this end, we analyzed the phosphorylation status of a universal tyrosine kinase substrate, the transforming Shc adapter protein, in fibroblasts expressing the viral oncogene. We report that the level of Shc phosphorylation does not decrease in PyLT-expressing fibroblasts after growth factor withdrawal and that this PyLT-mediated effect does not require interaction with protein encoded by the retinoblastoma susceptibility gene. We also found that the chronic activation of the adapter protein is correlated with the binding of Shc to Grb-2 and with defects in the downregulation of mitogen-activated protein kinases. In fibroblasts expressing the nuclear oncoprotein, we also observed the formation of a PyLT-Shc complex that might be involved in constitutive phosphorylation of the adapter protein. Viewed comprehensively, these results suggest that the cell cycle progression induced by PyLT may depend not only on the direct inactivation of nuclear antioncogene products but also on the indirect induction, through the alteration of cytoplasmic pathways, of growth factor-dependent nuclear signals.
PMCID: PMC103967  PMID: 9882348
3.  A Natural Hepatocyte Growth Factor/Scatter Factor Autocrine Loop in Myoblast Cells and the Effect of the Constitutive Met Kinase Activation on Myogenic Differentiation 
The Journal of Cell Biology  1997;137(5):1057-1068.
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.
PMCID: PMC2136220  PMID: 9166406
4.  The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts 
Molecular Biology of the Cell  1998;9(6):1449-1463.
It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.
PMCID: PMC25368  PMID: 9614186

Results 1-4 (4)