Modulation of p53 activities by the prolyl-isomerase PIN1 and the bromodomain protein BRD7

ABSTRACT: MODULATION OF p53 ACTIVITIES BY THE PROLYL-ISOMERASE PIN1 AND THE BROMODOMAIN PROTEIN BRD7 The tumour suppressor p53 belongs to a family of transcription factors that play key roles in maintaining genomic stability and cellular homeostasis. The orchestration of the appropriate cellular responses depends on the fine regulation of p53's functions through post-translational modifications and interaction with other proteins. In many years of intense study a considerable knowledge on p53 activity has been achieved, yet a greater insight is needed on the specificity of its response. In the first part of this thesis a novel mechanism in the regulation of p53-mediated apoptotic response has been disclosed. It has been demonstrated that upon severe stress signalling p53 dissociates from iASPP, an anti-apoptotic co-factor that inhibits p53 apoptotic functions, and that key roles in this process are played by the prolyl isomerase Pin1. Moreover, it emerged that phosphorylation at p53 Ser46 is required for Pin1-mediated dissociation of the p53-iASPP complex thus providing a mechanistic explanation for the relevance of this site in p53 mediated apoptosis. Notably, the role of Pin1 in assisting the dissociation of p53 from iASPP appears to be independent from Pin1-induced acetylation of p53 and dissociation from Mdm2, further confirming that Pin1 may modulate p53 activity at different levels. A different approach to gain insight on the mechanisms governing p53 regulation is the analysis of p53 protein interaction profiles. The bromodomain containing protein Brd7 was identified as a common interactor of the p53 family proteins in a yeast two hybrid screening conducted in our lab. The presence of the bromodomain and evidences from literature made Brd7 a promising candidate for modulating the p53 pathway at the transcriptional level. This protein and its functional interaction with p53 have been therefore characterized in the second part of this thesis. Upon depletion of Brd7 expression in cells it has been demonstrated that Brd7 is required for efficient cell-cycle arrest in U2OS cells upon challenging with genotoxic stimuli. This effect appeared to be due to a reduction in p21 expression that occurred upon Brd7 depletion and under stress condition. The down-regulation of p21 as a consequence of Brd7 silencing occurred at the transcriptional level and proved to be p53-depedent. Taken together the data reported in the second part of this thesis suggest a role for Brd7 as a positive regulator of p53 transcriptional activity during cell-cycle arrest response and that this function might be exerted by regulating p53-mediated transcription on a chromatin context. Further analysis is needed to dissct the role of this functional interaction. Yet, preliminary investigation suggest that Brd7 might be an important modulator of p53 response and that it can be an important means for p53 to crosstalk with other signalling pathway. Together, the data presented in this thesis contribute to achieve greater knowledge on the mechanism that govern p53 response. As the p53 pathway is compromised to some degree in almost all human cancers, this would be also of great relevance in designing new targeted strategies for cancer treatment.