Fig 2: Model of the regulatory function of the N-terminal specific peptides in TA*p63a. Models are shown explicitly for TA*p63a but represent GTAp63a as well.a In the first model, TA*p63a’s and GTAp63a’s specific N-termini bind to the same interaction site as the TAD (red) on the TD (cyan). Consequently, the dimeric conformation has an additional “safe lock”. In case of dissociation of the TAD, the TA*-GTA* peptide (blue-orange) is able to keep the protein in its dimeric state and the TAD in close proximity for rebinding. b In the second model, TA*p63a’s and GTAp63a’s specific peptides are binding in their inactive, dimeric state to a further interaction site independent of the TAD keeping p63 in a more tightly packed state by building a “double lock” safety mechanism. c Schematic representation of the activity difference between the two oligomeric states of TA*p63a (GTAp63a) and TAp63a. The dimeric conformation of TA*p63a shows a higher stability due to the additional stabilization of the N-terminal peptides in comparison to TAp63a. The open, tetrameric state of TA*p63a, however, possess at least four further binding sites for the co-activator p300, resulting in a greater transactivation potential. Consequently, the activity difference between the inactive and active state of TA*p63a (?TA*p63a, black) is higher than the one of TAp63a (?TAp63a, gray)

Fig 3: The kinases Chk2 and CK1 are able to phosphorylate and activate TA*p63a and GTAp63a like TAp63a, however, the high tetramer/dimer ratio of TAp63a cannot be reached.a, b BN-PAGE and phosphoshift SDS-PAGE analysis of wt and Chk2 priming site mutants (S582/S583A) followed by western blotting for myc-tagged TA*p63a and GTAp63a in comparison to TAp63a. Three hundred nanogram expression vector carrying the p63a gene were transiently transfected in U2OS cells (12-well plate). The next day, cells were treated with 10 µM Dox for 6 h. Migration of the different oligomeric states is indicated by T (tetramer), D (dimer), and M (monomer). The upper and lower panels in b show the same data with additional contrast enhancement in the lower panel. c BN-PAGE and phosphoshift SDS-PAGE analysis of H1299 cells lysate stably expressing either myc-tagged TAp63a, TA*p63a, or GTAp63a. Western blot analysis was carried out using an anti-myc antibody (4A6, Merck). Cells were pre-incubated with increasing Chk2 (BML-277, Merck) or CK1 inhibitor (PF670462, Sigma–Aldrich) concentrations (5, 25, 50 µM) for 1 h. Afterwards, 10 µM Dox was added and cells were harvested 6 h later. Migration of the different oligomeric states is indicated by T (tetramer), D (dimer), and M (monomer). Vinculin was used as loading control. Protein levels loaded on BN-PAGE were adjusted to equal p63 amounts by prior western blot analysis. SDS-PAGE to detect phosphoshifts were not input adjusted

Fig 4: Exons encoding the N-terminal p63 isoforms, domain architecture as well as oligomeric conformation of p63 isoforms.a Testis specific GTAp63 is encoded by an upstream exon, named U1, originating from retroviral LTR insertion. mRNA splicing fuses these exons directly to the exon 2, on which the GTA*peptide and the N-terminal part of TAD are located, in this way skipping exon 1. TA*p63 is encoded on exon1. Main splicing events are illustrated by solid lines, less frequently splicing by dotted lines, start codons are indicated by arrows. b Amino acid sequences of the N-terminal p63 isoforms of the corresponding exons, start codons are indicated by arrows. c Domain architecture and oligomeric conformation of p63 isoforms. GTAp63 and TA*p63 possess an N-terminal elongation compared to TAp63, comprising the TA*- or GTA-peptide, respectively, and the common GTA*-peptide, whereas the residual domains are shared by all three isoforms. ?Np63 possess a truncated transactivation domain. The C-terminal ß- and ?-isoforms are created via alternatively splicing. Only the inactive state of TAp63a has a dimeric oligomeric conformation, all other TAp63 and ?Np63 isoforms show a tetrameric conformation

Fig 5: Transactivation potential of constitutively tetrameric TA*p63 and GTAp63 isoforms and its correlation with p300 Taz2 domain interaction.a TA assay of C-terminal TA*p63 and GTAp63 isoforms in comparison to TAp63 C-terminal isoforms on the p21 promotor. p53 wt was used as positive, the p53 R175H mutant as negative control. Hundred nanogram of each plasmid (pcDNA3, pGL3 and pRL-CMV) were transiently transfected in Saos-2 cells (12-well plate). Cells were harvested 24 h after transfection and assay was performed. b, c TA titration assay of TA*p63? and GTAp63? compared to TAp63? on the p21 promotor. Hundred nanogram of pGL3 and pRL-CMV and increasing amount of p63 DNA (10 ng, 25 ng, 50 ng, 100 ng, 150 ng) were transiently transfected in Saos-2 cells. Empty vector was added to a total amount of 350 ng DNA for transfection (12-well plate). Cells were harvested 24 h after transfection and assay was performed. Protein levels were determined via western blotting. Fold induction is indicated with white bars, relative protein level with blue colored bars. The protein level of the highest DNA amount of TAp63a was set to 1. Dots represent protein level of each biological triplicate. (d) SDS-PAGE followed by western blotting for myc-tagged TAp63?, TA*p63? and GTAp63? protein level in the TA titration assay performed on p21 promotor in Saos-2 cells. GAPDH was used as loading control. p63 isoforms were detected with the p63 antibody ab124762 (Abcam) for sensitivity reasons. e TA assay of FWL and potential new TA motif mutants to alanine in the N-terminus of the TA*/GTAp63 isoforms (TA*p63: Y18* F22*, W31* Y35*; GTAp63:.W9' F12' V15', W29' Y33') on the p21 promotor. p53 wt was used as positive control, the p53 R175H mutant as negative control. Hundred nanogram of each plasmid (pcDNA3, pGL3 and pRL-CMV) was transiently transfected in Saos-2 cells (12-well plate). Cells were harvested 24 h after transfection and assay was performed. Bars represent the mean value of the biological triplicate, error bars represent SD, crosses represent mean value of the technical replicates. f Fluorescence anisotropy experiment with the Taz2 domain of p300 and either N-terminal fluorescein-tagged TA*-GTA*- (1*-39*) or GTA-GTA*- (1'-37') peptide. A peptide concentration of 100 nM was used for each measuring point with increasing concentration of p300 Taz2 domain