Ignacio Nojek - Research Associate

The p53 transcription factor protects cells from malignant transformation, and the development of most tumors is associated with loss of p53 function. The activated p53 tumor suppressor elicits apoptosis, cell cycle arrest and senescence in response to a variety of forms of cellular stress, including DNA damage, oncogene hyperactivation and hypoxia, thereby preventing the formation of tumors.  The p63 gene is a recently discovered member of the p53 gene family sharing 63% homology in its DNA-binding domain. p63 has six different isoforms, which elicit different biological responses. The transactivation (TA) isoforms alpha, beta and gamma are capable of transactivating a set of target genes, some of which overlap with targets downstream of p53. The ΔN p63 isoforms alpha, beta and gamma, produced from an intronic promoter, contain the same DNA-binding and oligomerization domain as the TA isoforms but lack the transactivation domain, located in the N-terminal portion of the protein, and thus, can act as a dominant-negative inhibitors of p53. Despite their structural homology, the p53 family members have distinctive biological functions. While p53 is a key gatekeeper for genomic stability, p63 is crucial during development and differentiation. p63 appears to be essential in epithelial and limb development, and mutations in this gene have been found in several human ectodermal syndromes involving limb development and ectodermal dysplasia, including ectrodactyly, ectodermal dysplasia and cleft lip/palate (EEC) syndrome; nonsyndromic split hand/foot malformation (SHFM); ankyloblepharon, ectodermal dysplasia, clefting (AEC) syndrome; acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome; and limb-mammary syndrome (LMS). On the other hand, recent genome-wide analysis has revealed that TAp63 activates various apoptotic genes previously characterized to be p53 targets. Furthermore, gene-disruption experiments and ChIP analyses indicate that p63 gene is fully re    quired for p53 dependent activation of specific apoptotic genes.

Aims and Experimental Approaches:
The main objective of my project is to elucidate the mechanism of synergistic and/or antagonistic transcriptional regulation by p53 and p63 isoforms. I began to study this issue by several approaches. First, I am studying the DNA-binding properties of different p63 isoforms by in vitro DNAse I footprinting assays and in vivo Chromatin Immunoprecipitation (ChIP). In order to perform the in vivo ChIP studies we need to score the binding of p63 to different gene promoters on different cell lines that express p63 endogenously. It has been found that squamous cell carcinomas express high levels of the ΔN-p63 isoform, so our initial efforts are mainly focused on those specific cell lines, as well as others that express p53 as a counterpart. Second, I am searching for factors that interact with the sterile-alpha motif (SAM) of p63, a known protein-protein interaction domain that is commonly mutated in the aforementioned syndromes. I am doing this by GST-pull-down assays from nuclear extracts, stable expression and purification of Flag-tagged p63 isoforms and yeast two-hybrid assays.