Sirico, Anna (2014) DISSECTING THE MOLECULAR MECHANISMS AT THE BASIS OF IMPAIRED p63 TRANSCRIPTIONAL ACTIVITY IN AEC SYNDROME. [Tesi di dottorato]

Anteprima

Testo tesi e paper.pdf Download (2MB) | Anteprima

Abstract

AEC (Ankyloblepharon- Ectodermal defects- Cleft lip/palate syndrome) syndrome is an autosomal dominant disorder mainly characterized by ectodermal dysplasia, skin erosions and cleft lip and/or palate. This disorder is caused by missense mutations in the Sterile Alpha Motif (SAM) domain and frameshift mutations in the Post-SAM (PS) domain of the transcription factor p63, a crucial regulator of embryonic development of stratified epithelia. To fully understand the molecular mechanisms associated with the pathogenesis of AEC syndrome, we analyzed transcription ability and DNA binding capacity of AEC causative p63 mutant proteins in heterologous system. L514F, G534V, C519R, D544Y, mutations involving SAM domain, and E570fsX94 and N620fsX44, mutations in the PS domain, showed an impaired transactivation ability consequent to a reduced DNA binding capacity, both when transfected alone and in combination with wild-type protein. To further explore the role of the two C-terminal domains of p63alpha isoform, that are involved in AEC syndrome, we obtained two deletion p63 mutants that lack alternatively SAM (p63DSAM) or PS (p63DPS) domains, and tested transactivation and DNA binding. We found that the SAM domain, but not the PS one, is strongly involved in these functions, since the deletion of the whole domain impaired transactivation and DNA binding ability, in spite of an intact DNA binding domain of p63. To test the physiological significance of our findings, we analyzed the effect of an endogenous L514F p63 mutant in primary keratinocytes. To this aim we took advantage of a conditional knock-in mouse model (p63+/floxL514F) recently generated in our laboratory. In accordance with previous results, also in its natural context AEC p63 mutant showed a reduced DNA binding and decresed expression levels of different p63 target genes, such as Fgfr2, IRF6, Dsc3, Dsp, Krt5 and Krt14, both in homozygous and in heterozygous mutant keratinocytes. Finally, we tested the effects of L514F p63 mutant in vivo mating p63+/- mice with knock-in p63+/L514F ones, previously generated in our laboratory that closely resembles the human disease but died soon after birth for cleft palate, to obtain p63-/L514F mice. At E18.5 we observed epidermal, limbs and craniofacial development defects and with ChIP assays on E14.5 embryos skin an impairement in DNA binding ability in p63-/L514F mice was confirmed. These results shed light on the mechanisms underlying AEC syndrome, since all the analyzed mutant p63 proteins show an impairment in DNA binding ability, moreover this reduction is observed in heterozygous keratinocytes suggesting that L514F p63mutant acts in a dominant negative manner, forming with wild-typeproteins tetramers not well working. Using p63 deletion mutants wedemonstrate that an intact SAM domain is required for transactivationand DNA binding ability in DNp63alpha context, while large rearrangements of PS domain, such as frameshift mutations but not whole domain deletion, show same effects, suggesting that this domain is not required for the described p63 functions. Finally, with in vivo experiments we demonstrate the crucial role of alpha-isoform of p63 in epidermal, limbs and craniofacial development and that its mutations, although in the presence of intact  and  isoforms, strongly impaired development and DNA binding capacity, which explains pathogenesis of AEC syndrome.

Downloads

Actions (login required)

Modifica documento