Volume 2, Issue 3, Pages 305-316 (September 1998) p63, a p53 Homolog at 3q27–29, Encodes Multiple Products with Transactivating, Death-Inducing, and Dominant-Negative Activities  Annie Yang, Mourad Kaghad, Yunmei Wang, Emily Gillett, Mark D Fleming, Volker Dötsch, Nancy C Andrews, Daniel Caput, Frank McKeon  Molecular Cell  Volume 2, Issue 3, Pages 305-316 (September 1998) DOI: 10.1016/S1097-2765(00)80275-0

Figure 1 Primary Structure Alignments of p53, p73, and p63 Human p53, human p73β, human TAp63γ, and murine TAp63γ are presented, with residues identical to p53 shaded in gray and remaining consensus residues shaded in black. Molecular Cell 1998 2, 305-316DOI: (10.1016/S1097-2765(00)80275-0)

Figure 2 Genomic Origin and Diversity of p63 Isotypes (A) Schematic of human p63 gene structure highlighting positions of exons (coding sequences in black), the two promoters in exon 1 (black arrow), and exon 3′ (gray arrow), and the major posttranscriptional splicing events that give rise to the major p63 isotypes. (B) Domain structure of p53, p73α and β, and the major p63 isotypes, TAp63α, β, and γ, and ΔNp63 α, β, and γ, highlighting regions involved in transactivation (TA), DNA binding, and oligomerization (oligo). White box denotes 39 aa N-terminal extension unique to TA*p63. Gray box represents 14 aa unique to ΔN-p63. (C) Sequence alignment of N termini of murine and human p63 including that found in TA*p63, TAp63, and ΔNp63. (D) Alignment and comparison of the human p63α, β, and γ C-terminal sequences. Molecular Cell 1998 2, 305-316DOI: (10.1016/S1097-2765(00)80275-0)

Figure 3 Chromosomal Localization of Human and Mouse p63 Gene (A) Schematic of chromosome 3 showing localization of human p63 gene at 3q27–29 based on fluorescence in situ hybridization with a p63 genomic PAC clone. (B) Schematic of the proximal end of mouse chromosome 16 showing the location of murine p63 gene, as determined by linkage analysis against Jackson Laboratory interspecific backcross panels BBS and BSB. Loci mapping to similar positions are presented in alphabetical order, and missing typing is inferred from surrounding data where assignment was unambiguous. Molecular Cell 1998 2, 305-316DOI: (10.1016/S1097-2765(00)80275-0)

Figure 4 Immunolocalization of p63 in Human Epithelial Tissues Paraffin sections of normal human epithelial tissues probed with monoclonal antibodies to p63 using an alkaline phosphatase reporter system. (A) p63 staining in foreskin showing nuclear localization of p63 in basal epithelial cells. (B) p63 localization to basal cells of ectocervical epithelium. (C) p63 localization in basal cells of vaginal epithelium. (D) p63 staining of basal cells of urothelium. (E) p63 staining of epithelial cell layer below luminal cells in prostate. Molecular Cell 1998 2, 305-316DOI: (10.1016/S1097-2765(00)80275-0)

Figure 5 Tissue Distribution of p63 Isotypes (A) RT-PCR analysis of total RNA prepared from various adult mouse tissues using oligonucleotide primers designed to amplify TAp63 isotypes, revealing an ∼410 bp product in heart, testis, kidney/adrenal, thymus, brain, and cerebellum. (B) RT-PCR analysis using template RNA in (A) with oligonucleotide primers designed to yield an ∼240 bp product for ΔNp63 isotypes, revealing expected product in kidney/adrenal, spleen, and thymus. (C) Gel electrophoresis of RNA used as template in RT-PCR analyses to determine template integrity. (D) Analysis of p63 transcripts in human epithelial tissues. RT-PCR analyses of RNA from primary human foreskin keratinocytes, ectocervical cells, and the human cervical carcinoma cell line ME180 using oligonucleotides designed to amplify TAp63 transcripts (left panel) and ΔNp63 transcripts (right panel). The ME180 cells show products corresponding to both the TAp63 and the ΔNp63 transcripts, while RNA from primary keratinocytes and ectocervical cells yield predominantly products from ΔNp63 transcripts. (E) Western blot of primary human foreskin keratinocytes (1°HFK), ME180 human cervical carcinoma cells (ME180), and BHK cells expressing epitope-tagged p63 isotypes (TA*p63γ, TAp63γ, ΔNp63γ, TA*p63α, TAp63α, and ΔNp63α) using the 4A4 anti-p63 monoclonal antibody. The major p63 species in primary keratinocytes migrates slightly faster than the epitope-tagged ΔNp63α protein. Molecular Cell 1998 2, 305-316DOI: (10.1016/S1097-2765(00)80275-0)

Figure 6 Transactivation of p53 Reporter Genes by p63 Isotypes Transcriptional activation of p53 reporter gene in Saos-2 cells transfected with the indicated p53 and p63 expression constructs. Chemiluminescence signal from reporter β-galactosidase assays was normalized for transfection efficiency using cotransfected luciferase vector. Error bars indicate standard deviation in triplicate assays. Molecular Cell 1998 2, 305-316DOI: (10.1016/S1097-2765(00)80275-0)

Figure 7 Induction of Apoptosis by p63 Isotypes BHK cells transfected with identical amounts of wild-type p53 (A), mutant p53 (B), TAp63γ (C), ΔNp63γ (D), and ΔNp63α (E) were processed for immunofluorescence after 16 hr using epitope-tagged antibodies (left panel) and Hoechst dye for DNA staining (right panel). Wild-type p53- and TAp63γ-expressing cells showed high levels of apoptosis (arrowheads) despite very low protein expression, while ΔNp63γ yielded high protein expression and modest levels of apoptosis. Mutant p53 and ΔNp63α showed high levels of protein expression but control levels of apoptosis. Molecular Cell 1998 2, 305-316DOI: (10.1016/S1097-2765(00)80275-0)

Figure 8 Interactions among p63 Isotypes and p53 in Transactivation Assays (A) Transactivation analysis in Saos-2 cells transfected with a constant amount of wild-type p53 expression vector, minimal p53-reporter construct, and either ΔNp63γ or ΔNp63α expression vectors at ratios of 1:5 or 1:1 with respect to p53, as indicated. (B) Transactivation analysis in Saos-2 cells transfected with a constant amount of TAp63γ expression vector, p53 reporter construct, and either ΔNp63γ or ΔNp63α expression vectors at ratios of 1:5 or 1:1 with respect to TAp63γ, as indicated. Molecular Cell 1998 2, 305-316DOI: (10.1016/S1097-2765(00)80275-0)