ABSTRACT
The cdk inhibitor p57(kip2) plays a critical role in many differentiation processes by performing not only redundant but also specific functions. Compared to other cdk inhibitors, p57(kip2) shows a more restricted expression pattern during development and in adult tissues. We have previously reported that in muscle cells, p57(kip2) is induced by the myogenic factor MyoD through an indirect mechanism involving p73 proteins as intermediaries. We have also reported that p57(kip2) shows a differential responsiveness to MyoD-dependent regulation in different cell types. In this work we have further investigated the molecular mechanism by which MyoD activates p57 promoter. We show that the minimal promoter element able to confer MyoD responsiveness contains multiple Sp1 and Egr1 recognition sites and that both transcription factors are necessary for the increase in p57 RNA. We also suggest that the role of MyoD-induced p73 consists in promoting the binding of Sp1 to p57(kip2) promoter. Moreover, we show that Egr1 and Sp1 are concomitantly recruited to p57 promoter in vivo only in differentiation conditions and only in responsive cells. Bisulfite sequencing suggested a functional link between the methylation status and the differential activity of p57 promoter, both during differentiation and in distinct cell types. These results, which highlight the involvement of epigenetic factors in the regulation of p57 expression in muscle cells, could be of general relevance to explain its tissue and cell type restriction during development.
Subject(s)
Cell Differentiation/physiology , Cyclin-Dependent Kinase Inhibitor p57/metabolism , DNA Methylation , Gene Expression Regulation , Muscle, Skeletal/physiology , Protein Kinase Inhibitors/metabolism , Sp1 Transcription Factor/metabolism , Animals , Base Sequence , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p57/genetics , Fibroblasts/cytology , Fibroblasts/physiology , Humans , Mice , Mice, Knockout , Molecular Sequence Data , MyoD Protein/metabolism , Promoter Regions, Genetic , Sp1 Transcription Factor/geneticsABSTRACT
The cyclin-dependent-kinase inhibitors p21 and p57 are highly expressed in skeletal muscle where they redundantly control cell cycle arrest during differentiation. We have previously shown that p57 is a target of the myogenic factor MyoD in cells lacking p21. Here we show that MyoD induces p57 at the transcriptional level through a mechanism different from that involved in p21 regulation, since it is E-box-independent and requires new synthesized protein(s). We have identified p73 family members as the factors that mediate the activation of p57 through a 165bp promoter region. The levels of p73 alpha, beta and delta isoforms increase during muscle differentiation both in MyoD-expressing fibroblasts and in spontaneously differentiating C2 myoblasts. Moreover, the expression of a p73 dominant negative mutant interferes with the induction of p57. Finally, each of the isoforms up-regulated by MyoD, even when over-expressed alone, is capable of inducing p57 in p21-lacking fibroblasts. In contrast, the same p73 isoforms, either induced by MyoD or exogenously over-expressed, are unable to activate the expression of p57 in p21-expressing fibroblasts. Our finding that a transfected p57 promoter-reporter construct, unlike the endogenous gene, is responsive to both MyoD and p73 even in these cells, suggests that a cis-acting mechanism, probably involving a repressive chromatin structure, prevents the induction of p57 in p21-expressing fibroblasts.
