Figure 5: The CCAAT element mediates ΔNp63α- and E2F-1-dependent stimulation of the ATM promoter. (A) Schematic representation of the human ATM promoter organisation (Genebank U82828), showing the position of putative response elements which are mutated in this study. (B) The CCAAT sequence mediates ΔNp63α and E2F-1 stimulated ATM transcription. H1299 cells were co-transfected with 1 μg ΔNp63α respective with 1 μg E2F-1, 1 μg ATM-LUC (wt or CCAAT mutant) and 0.2 μg pRL-CMV control plasmids, then lysed and processed after 24 hrs. Specific ATM-LUC activity was determined as described in Figure 4 throughout, and data is normalised to wild-type reporter activity. (C) ΔNp63α and E2F-1 have additive effects on ATM promoter stimulation. H1299 cells were transfected with 0.5 μg ΔNp63α, E2F-1 or 0.5 μg each plasmid along with 1 μg ATM-Luc (wt or mutant) and 0.2 μg pRL-CMV control plasmids, and cells were harvested and processed after 24 hrs. Total DNA was balanced with empty pCDNA3.1 vector.

Mentions: The DNA binding (DB) domain of p63 is 65% homologous to that of p53, and p63 variants can recognise p53 response elements (REs) [2,22]. Thus, ΔNp63α-mediated ATM transcription could either involve direct binding to the ATM promoter via the DB domain, or indirect binding via an unidentified cofactor. Previous analysis of the ATM promoter failed to identify a p53 RE, although binding sites for several other transcription factors were identified [23]. We therefore used a series of ATM reporter constructs containing point mutations at putative REs to identify sequences required for basal and ΔNp63α-stimulated ATM transcription (see Figure 5A) [23]. Basal ATM reporter activity is absent in mutant ATM reporter constructs lacking either the Ire2 or Fse REs, which were previously found to control ATM transcription in cycling normal human fibroblast and lymphoblastoid cells [23]. ΔNp63α-induced ATM reporter activity was specifically attenuated by mutation of the NF-1 RE, encoded by the AGCCAAT sequence (Figure 5B), and containing a CCAAT element. Mutation of the CCAAT element also blocked E2F-1-mediated ATM promoter stimulation (Figure 5B). This was unexpected as deletion mutagenesis had previously located the E2F-1 target region to between -436 and -392 (Figure 5A 10379-10423), containing two putative E2F-1 REs. However, as this deletion also removes the CCAAT element at -435 - -429 (10380-10386), we reinterpret the original data to indicate that E2F-1 regulates ATM transcription through interaction with the same CCAAT element as ΔNp63α. In support of this, we found that ΔNp63α and E2F-1 do not synergistically activate the ATM promoter, but instead have additive effects (Figure 5C).

Craig AL, Holcakova J, Finlan LE, Nekulova M, Hrstka R, Gueven N, DiRenzo J, Smith G, Hupp TR, Vojtesek B - Mol. Cancer (2010)

Bottom Line: DeltaNp63alpha depletion by RNAi reduces steady-state ATM mRNA and protein levels, and attenuates p53 Serine-15 phosphorylation.Structure-function analysis revealed that the DeltaNp63-specific TA2 transactivation domain mediates ATM transcription in coordination with the DNA binding and SAM domains.Germline p63 point mutations are associated with a range of ectodermal developmental disorders, and targeted p63 deletion in the skin causes premature ageing.The DeltaNp63alpha-ATM-p53 damage-response pathway may therefore function in epithelial development, carcinogenesis and the ageing processes.

Affiliation: Cell Signalling Unit, Cancer Research Center, Western General Hospital, University of Edinburgh, Edinburgh EH4 2XR, UK.

Abstract: DeltaNp63alpha is an epithelial progenitor cell marker that maintains epidermal stem cell self-renewal capacity. Previous studies revealed that UV-damage induced p53 phosphorylation is confined to DeltaNp63alpha-positive cells in the basal layer of human epithelium.We now report that phosphorylation of the p53 tumour suppressor is positively regulated by DeltaNp63alpha in immortalised human keratinocytes. DeltaNp63alpha depletion by RNAi reduces steady-state ATM mRNA and protein levels, and attenuates p53 Serine-15 phosphorylation. Conversely, ectopic expression of DeltaNp63alpha in p63- tumour cells stimulates ATM transcription and p53 Serine-15 phosphorylation. We show that ATM is a direct DeltaNp63alpha transcriptional target and that the DeltaNp63alpha response element localizes to the ATM promoter CCAAT sequence. Structure-function analysis revealed that the DeltaNp63-specific TA2 transactivation domain mediates ATM transcription in coordination with the DNA binding and SAM domains.Germline p63 point mutations are associated with a range of ectodermal developmental disorders, and targeted p63 deletion in the skin causes premature ageing. The DeltaNp63alpha-ATM-p53 damage-response pathway may therefore function in epithelial development, carcinogenesis and the ageing processes.