The transcription factor p63 is a key regulator of ectodermal, orofacial and limb development. In particular, it plays a critical role in epithelial biology, contributing to development and maintenance of the stratified epidermis (1). Dominant mutations in the p63 gene are causative of several human hereditary syndromes, such as AEC, EEC, LMS and SHFM-IV (2). Unequivocal establishment of the role of p63 in the pathogenesis of these human hereditary syndromes is complicated by the fact that this protein exists in multiple isoforms with different, often contradictory, biological activities. Moreover, the interrelationships between specific p63 isoforms and other members of the family (p53 and p73) play an essential role into the proliferation and differentiation programme of developing cells. Despite increasing knowledge about the biological function of p63 in the tissues in which is expressed, relatively little is known about the mechanisms governing the expression levels of the p63 proteins. The function played by p63 in ectodermal differentiation and stratified epithelial progenitor-cell maintenance is well assessed, however, further studies are required to determine the interplay of p63 with other signalling pathways including those regulating the other p53 family members.
It is likely that interrelationships between p53 and specific p63 isoforms play an essential role into the proliferation and differentiation programme of developing cells. A current opinion is that p63 and/or p73 reside with p53 in larger transcriptional complex in which each sibling may regulate the activity of the others (3). To this respect, mutations affecting the relative stability of specific isoforms might grossly alter the fine tuning of p53 and p63 activity on specific promoters.
It has already been demonstrated that one component of the principal p53 regulatory pathway, the p300 acetylase, is clearly involved also in the control of p73 isoforms (4, 5) with very little being known about its role on p63 isoforms (6).
We are now interested in unravelling the role of the p300 acetylase on p63 regulation during keratynocites differentiation and during mouse limb development.
The p300 acetylase has a critical role in regulating p53 and p73 protein stability and transcriptional activity. Recently, a new p300 site has been described in p53, lysine 164, which integrity is essential for p53 activities (7) that is perfectly conserved in p63 and is found mutated to glutamic acid (K193E) in patient affected by the SHFM-IV syndrome.
Interestingly, we have made preliminary observations suggesting that the effects of p300 on p63 stability could be affected by this natural mutation.

We intend now to better investigate the mechanisms through which p300 regulates p63 expression since, depending on their relative expression levels, p300 can mediate either p63 protein stabilization or p63 protein degradation. This dual role of p300 has already been described for the p53 protein (8).
Our studies should contribute to the identification of the molecular mechanisms and the cellular players involved in the control of epithelial and limb development and homeostasis.
Specific aims and workplan

The main objective of this Project is to investigate the role of the p300 protein and the molecular mechanisms with which it controls p63 protein stability, to understand how the physiological turnover of wild type and human syndromes-derived mutant p63 isoforms is regulated/deregulated, during mouse limb development and keratynocites differentiation, using different cell lines and primary and/or stabilized keratinocytes.

References
1) Barbieri CE, et al (2006) p63 and epithelial biology. Exp Cell Res 312, 695-706
2) Brunner HG, et al (2002) P63 gene mutations and human developmental syndromes. Am J Med Genet 112, 284-290
3) King KE, Weinberg WC.(2007) p63: defining roles in morphogenesis, homeostasis, and neoplasia of the epidermis. Mol Carcinog 46, 716-24
4)Barlev NA, Hiu L, Chehab NH, Mansfield K, Harris KG, Halazonetis TD and Berger SL. Acetylation of p53 Activates Transcrition trough Recruitment of Coactivators/Histone AcetylTransferase. Molecular Cell 2001; 8: 1243-1254
5) Mantovani F, Piazza S, Gostissa M, Stano S, Zacchi P, Mantovani R, Blandinoo G and Del Sal G. Pin1 Links the Activities of c-Abl and p300 in regulating p73 Function. Molecular Cell 2004; 14:625-636
6) MacPartlin M, Zeng S, Lee H, Stauffers D, Jin Y, Thayers M and Lu H. p300 regulates p63 Trasc-riptional Activity. The Journal of Biological Chemistry 2005; 280: 30604-30610.
7) Tang Y, Zhao W, Chen Y, Zhao Y and Gu W. Acetylation Is Indispensable for p53 Activation. Cell 2008; 133:612-626
8) Grossman SR, Deato ME, Brignone C, Man Chan H, Kung AL, Tagami H, Nakatani Y and Livingston DM. Polyubiquitination of p53 by a ubiquitin Ligase activity of p300. Science 2008; 300:342-344.

The p63 transcription factor, homolog to the p53 tumor suppressor, is involved in development, cell differentiation, tumor suppression and response to genotoxic stress. Expression of the p63 protein is essential for normal development of the limbs and all stratified ephitelia. p63 Knock-Out (KO) mice die of dehydration after birth with truncations of the limbs, no epidermis and craniofacial defects reflecting ectodermal stem cells loss.
p63 mutations are found in several human syndromes, including the Acro Dermato Ungueal Lacrimal Tooth (ADULT) syndrome. ADULT patients display exfoliative dermatitis of the digits (1). Two ADULT patients, carrying the p63 mutation R298Q, display Ectodermal Dysplasia (ED) and Paroxysmal supraventricular tachycardia: this disturbance, in the ADULT patients, could be a consequence of atriomegalia resulting from inadequate cell adherance mediated by desmosomes as observed in Arhytmogenic Right Ventricular Cardiomiophaty (ARVC) patients.
p63 plays a role in the assembly of desmosomal adhesive complexes and in enhancing the ability of diferrent tissues, including the cardiac tissue, to resist to mechanical stresses (2).
Cardiac defects have been observed in the c-Abl Knock Out (KO) mice. cAbl is an ubiquitously expressed nonreceptor tyrosine kinase that, upon DNA damage, affects p63 protein stability leading to activation of proapoptotic genes. c-Abl KO newborn mice, display dramatically enlarged heart that is the primary cause of perintal death (3).
Our preliminary data show that in the HaCaT human keratinocyte cell line, expressing endogenous DNp63a and c-Abl, silencing of cAbl expression results in reduction of the p63 protein levels, indicating an interplay between p63 and c-Abl. In the H1299 and HaCaT cell lines, p63 overexpression activates transcription of the endogenous c-Abl gene suggesting the existence of a positive feedback loop between p63 and c-Abl. Moreover, a c-Abl promoter reporter plasmid was activated by overexpression of wild type Dnp63alpha while, interestingly, mutant p63 protein had impaired transcriptional activation capacity on this promoter.
These observations point to feed-back loop acting on p63 and c-Abl and we want to verify if this loop has a physiological role in heart development.
Reference

1. Am J Med Genet 112 284-90 2002
2. Cell 120 843-56 2005
3. PNAS 3 1136-41 2010