Characterization of the c-Myb-responsive region and regulation of the human type I collagen alpha 2 chain gene by c-Myb

We have characterized the role of c-Myb and B-Myb in the regulation of human type I collagen alpha2 chain gene expression in fibroblastic cells. We have identified four Myb-binding sites (MBSs) in the promoter. Transactivation assays on wild type and mutant promoter-reporter constructs demonstrated that c-Myb, but not B-Myb, can transactivate the human type I collagen alpha 2 chain gene promoter via the MBS-containing region. Electrophoretic mobility shift assay experiments showed that c-Myb specifically binds to each of the four MBS; however, the mutagenesis of site MBS-4 completely inhibited transactivation by c-Myb, at least in the full-length promoter. In agreement with these results, c-myb(-/-) mouse embryo fibroblasts (MEFs) showed a selective lack of expression of type I collagen alpha 2 chain gene but maintained the expression of fibronectin and type III collagen. Furthermore, transforming growth factor-beta induced type I collagen alpha 2 chain gene expression in c-myb(-/-) MEFs, implying that the transforming growth factor-beta signaling pathway is maintained and that the absence of COL1A2 gene expression in c-myb(-/-) MEFs is a direct consequence of the lack of c-Myb. The demonstration of the importance of c-Myb in the regulation of the type I collagen alpha 2 chain gene suggests that uncontrolled expression of c-Myb could be an underlying mechanism in the pathogenesis of several fibrotic disorders.     

Inhibition of Crm1-p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation

Poly(ADP-ribose) polymerase 1 (PARP-1) and p53 are two key proteins in the DNA-damage response. Although PARP-1 is known to poly(ADP-ribosyl)ate p53, the role of this modification remains elusive. Here, we identify the major poly(ADP-ribosyl)ated sites of p53 by PARP-1 and find that PARP-1-mediated poly(ADP-ribosyl)ation blocks the interaction between p53 and the nuclear export receptor Crm1, resulting in nuclear accumulation of p53. These findings molecularly link PARP-1 and p53 in the DNA-damage response, providing the mechanism for how p53 accumulates in the nucleus in response to DNA damage. PARP-1 becomes super-activated by binding to damaged DNA, which in turn poly(ADP-ribosyl)ates p53. The nuclear export machinery is unable to target poly(ADP-ribosyl)ated p53, promoting accumulation of p53 in the nucleus where p53 exerts its transactivational function.     

Central and carboxy-terminal regions of human p53 protein are essential for interaction and complex formation with PARP-1

It has been previously described by different groups that poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53 form tight complexes. We investigated which domains of human PARP-1 and of human wild-type p53 were involved in this protein-protein interaction. We generated baculoviral constructs encoding full length protein or distinct functional domains of both proteins. Baculovirally expressed wild-type p53 was posttranslationally modified. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 were sufficient to confer binding to PARP-1. The amino-terminal part harboring the transactivation functional domain of p53 was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were necessary for complex formation with p53 protein. Finally, we explored the functional significance of the interaction between both proteins. Inactivation of PARP-1 resulted in the reduction of p53 steady-state levels. Inhibition of nuclear export by leptomycin B prevented accelerated degradation of p53 in PARP-1 KO cells and led to accumulation of p53 protein. Considering the fact that the accelerated p53 nuclear export in the absence of PARP-1 contributes to enhanced p53 degradation, we conclude that PARP-1 may mask the NES of p53 through complex formation with its carboxy-terminal part, thereby preventing the export.