p53 is the primary mediator responsible for removing DNA damaged epidermal cells , and p53 phosphorylation at the CK2-site is required to suppress UV-induced skin cancer development in mice . We previously reported the striking confinement of UV-induced p53 phosphorylation at the key damage-response CK2 and ATM sites to ΔNp63α-positive basal skin cells, despite substantial p53 stabilization throughout the epidermis . We next aimed to identify novel factors that control damage-induced p53 phosphorylation in a keratinocyte model system, and discovered that the epithelial stem cell marker ΔNp63α is a novel ATM regulator that controls p53 Serine-15 phosphorylation through transcription of the ATM kinase. Loss of ΔNp63α by RNAi or differentiation reduced ATM-dependent phosphorylation and conversely, ΔNp63α overexpression stimulated ATM signaling. A recent genome-wide screen reporting that ATM expression is reduced by 30-60% in p63 siRNA-treated epithelial cell lines supports our finding .
Post-translational activation of the ATM kinase by ionizing radiation, oxidative stress, chemotherapeutic drugs  and UV radiation is well-established . However, ATM transcriptional regulation has been shown to occur both in vitro and in vivo . E2F-1 stimulation of ATM transcription  has been implicated in oncogene-mediated p53 activation . In contrast, epidermal growth factor sensitizes cells to ionizing radiation through Sp1-mediated repression of ATM transcription . We have shown that p63 ΔN-isotypes are novel positive regulators of ATM transcription that interact with the promoter CCAAT sequence. p63-dependent gene regulation has been reported to occur through interaction of the DB domain with a p53 RE . However, the lack of similarity of the CCAAT sequence to classical p53 REs suggests that p63 interaction with a CCAAT element is indirect, and requires a CCAAT-binding mediator. We show that the E2F-1 regulates ATM transcription through the same CCAAT sequence, not a canonical E2F-1 response element, suggesting that a CCAAT-binding cofactor integrates activation signals from diverse ATM transcriptional regulators. CCAAT-binding proteins include NF-1/CTF , NF-Y  and C/EBP . NF-Y can mediate ΔNp63α-dependent transcription in human keratinocytes [10, 36], p53-dependent repression of cell cycle genes , and transcriptional activation by p53 gain-of-function mutants . However, we found that coexpression of the NF-YA isoform inhibits ΔNp63α stimulation of the ATM reporter (data not shown), presumably by displacing the unidentified ΔNp63α coactivator from the ATM promoter CCAAT element. Ongoing studies aim to further delineate the mechanism of ΔNp63α-mediated ATM transcriptional control by identifying ΔNp63α binding partners in epithelial cells.
Based on our findings so far, cooperation of three distinct functional domains is required to mediate p63-dependent ATM transcription. We found that p63 ΔN-isotypes transcriptionally activate the ATM gene, whereas TA-isotypes do not, highlighting an essential role for the TA2 transactivation domain in mediating ΔNp63α function. Future studies will aim to determine which cofactors are recruited to this region, and whether their access is controlled by TA2 domain post-translational modification, similar to the p53 model . There was also a requirement for an intact p63 DB domain, despite the absence of a canonical p53 RE in the ATM promoter. However, in addition to providing a surface for the sequence-specific binding of DNA, the p53 DB domain modulates p53 function by providing a contact interface for regulatory proteins such as ASPP1, Mdm2, and DAPK superfamily kinases [40, 41], and the high degree of conservation of the p63 DB domain suggests that a similar interface exists on p63. Finally, the p63 SAM domain forms a binding site for NF-Y , and SAM domain disease-associated mutants have decreased transcriptional repressor and activator function [7, 42]. We found that AEC point mutations within the SAM domain  inhibited ΔNp63α-stimulated ATM transcription and ATM-dependent p53 phosphorylation, indicating that this domain may be essential for cofactor recruitment by the ΔNp63α. Interestingly, the AEC clinical phenotype predominantly involves skin defects without associated limb abnormalities , consistent with a skin-specific role for ΔNp63α-ATM-p53 signaling in mediating normal ectodermal development. Therefore, the coordinated assembly of several cofactors may be required for fully functional p63 transcriptional machinery.
According to our model, elevated ΔNp63α-dependent ATM transcription primes p53 leading to damage-sensitivity in epithelial stem cells. Loss of p63-ATM-p53 pathway function will compromise epithelial stem cell function and promote premature ageing or skin carcinogenesis. Interestingly, transgenic mice with a specific p63-deficiency in the epithelium show increased senescence and an accelerated ageing phenotype . Although transgenic mice lacking the Serine-18 (equivalent to human Serine-15) ATM phosphorylation site are not cancer-prone , it is now important to determine whether mutation at p53 Serine-18 enhances sensitivity to UV-induced skin tumorigenesis, similar to mutation of the CK2-site. Interestingly, p53S18A/S23A (ATM-/CHK2-sites) double mutant mice develop a spectrum of spontaneous tumours distinct from p53S23A and p53-null mice, and show accelerated skin ageing phenotypes when crossed into a repair-deficient background .
Further, activation of the ATM-CHK2 pathway during early tumorigenesis has been reported to provide a selective pressure for p53 mutation . The discovery that the ΔNp63 promoter is subject to both p53-mediated activation and repression by ΔNp63α , and that ATM-dependent phosphorylation mediates ΔNp63α degradation  suggests that activity of the damage-response ΔNp63α-ATM-p53 pathway is finely modulated by complex feedback mechanisms. Further dissection of this pathway should provide molecular targets for combating cancer and ageing.