p53, p63, and p73 genes are located on chromosomes 17p13.1, 3q27-29, and 1p36.2-3, respectively. These genes encode proteins with similar domain structures and significant amino acid sequence homology in the transactivation, DNA-binding and oligomerization domains (). The highest amino acid identity is in the DNA-binding domain (~60%). Evolutionally, this domain is the most conserved, suggesting that regulation of transcription plays a pivotal role in an array of functions attributed to the p53 family. Less similarity is found in the oligomerization and transactivation domains (~30%).
Figure 1 Architectures of human TP53, TP73, and TP63 genes. (A) TP53, TP73, and TP63 genes encode the transactivation (TAD), DNA-binding (DBD), and oligomerization (OD) domains. TP73 and TP63 encode additional SAM (Sterile Alpha Motif) domain. Percentage homology (more ...)
The founding member of the p53 family, the p53 protein, had been discovered more than three decades ago [12
]. For a long time, it had been assumed that p53 is expressed as a single polypeptide. However, when it had been found that the p63 and p73 genes encoded a large variety of diverse transcripts, the p53 gene transcription was revisited. Now we know that p53 forms multiple variants.
Transcriptions of p53, p63, and p73 genes are regulated by similar mechanisms. It is controlled by two promoters: P1 and P2, where P2 is an alternative intragenic promoter (). One study in silico
provided evidence for the existence of a third putative promoter in the first intron of human TP73
]. Therefore, it would not be surprising if additional gene promoters will be found in the future. An extensive alternative splicing adds further diversity to the promoters' products. The produced transcripts and proteins can be generally categorized into two main groups, termed TA and ΔN [15
]. TA variants contain the N-terminal transactivation domain while ΔN isoforms lack the entire (or part of) domain. It was initially thought that ΔN isoforms are only generated by the P2 promoter whereas the P1 promoter regulates TA isoforms. Further analysis of alternative mRNA splicing revealed that some transcriptionally deficient isoforms are products of the P1 promoter. For example, the P1 promoter of the TP73
gene regulates TAp73 isoforms and isoforms, which lack the TA domain: ΔEx2p73, ΔEx2/3p73, and ΔN′p73. The latter isoforms are missing either exon 2 (ΔEx2p73) or both exon 2 and 3 (ΔEx2/3p73) or contain an additional exon 3′ (ΔN′p73) [17
]. Other ΔNp73 transcripts are products of the P2 promoter. Similar to p73, the P1 promoter of the p53 gene produces transcriptionally active isoforms [5
]. The alternative splicing is responsible for transcriptionally deficient isoforms of Δ40p53, which missing the first 40 amino acids at the N-terminus [5
]. Additional p53 transcriptionally deficient isoforms (Δ133p53 and Δ160p53) are regulated by the P2 promoter located in intron 4 of the p53 gene [5
Additional diversity of p53, p63, and p73 transcripts is generated by alternative splicing at the 3′ end of the transcripts (). These splice variants are traditionally named with letters of the Greek alphabet. Initially, three such splice variants have been described for p63 and p53 (α
), and nine for p73 (α
, and η
]. Later, additional p63 splice variants (δ
) and p53 (δ
, ΔE6) were reported [26
]. However, it should be noted that a majority of p53, p63, and p73 studies focus on a few isoforms, primarily α
, and γ
. Little is known about the functions of other isoforms. The combination of alternative splicing at the 5′ and 3′ ends, alternative initiation of translation and alternative promoter usage can significantly increase protein diversity. For example, N-terminal variants (p53, Δ40p53, Δ133p53, and Δ160p53) can be produced in α
, and γ
]. Theoretically, the p53 gene can produce at least 20 isoforms, p63 at least 10, and p73 more than 40, though not all have been experimentally confirmed.
p53, TAp63, and TAp73 share significant functional resemblance. They can induce cell cycle arrest, apoptosis, or cellular senescence. This similarity can be explained, at least in part, by transactivation of the same transcriptional targets. Genome-wide analyses found an overlap of the transcription profiles of p53, TAp73, and TAp63, though unique targets were identified as well. Analyses using chromatin immunoprecipitation, reporter, and gel-shift assays found that TAp73 and TAp63 interact with p53-responsive elements.
The transactivation and apoptotic potential of p53, TAp73, and TAp63 vary greatly depending on the isoform. TAp63γ
are similar to that of p53α
]. Other isoforms are considered less active on the p53 target gene promoters [9
]. Some isoforms are characterized by a variation in domain structure. TAp73α
have an additional domain at the COOH-terminus that is not found in p53. This domain, termed SAM or Sterile Alpha Motif, is responsible for protein-protein interactions and is found in a diverse range of proteins that are involved in developmental regulation. It is also implicated in transcriptional repression [31
]. Beta and gamma isoforms of p53 are missing most of the oligomerization domain that results in decreased transcriptional activity [5
ΔN isoforms function as dominant-negative inhibitors of TA counterparts (). Promoter competition and heterocomplex formation have been suggested to explain this phenomenon [17
]. In the promoter competition mechanism, the suggestion is that ΔN competes off TA isoforms from their target gene promoters, thus preventing efficient transcription. In the heterocomplex formation mechanism, ΔN isoforms would inhibit TA by forming hetero-oligomeric complexes.
Interactions of p53 family isoforms. N-terminally truncated isoforms of p53, p73, and p63 play a dominant-negative role inhibiting transcriptional and other biological activities of TA isoforms.
ΔN isoforms of p53 and p73 are regulated by a negative feedback loop mechanism. Analogous mechanism was not described for p63 despite its significant similarity to p73. In a nutshell, TA isoforms are able to induce transcription of ΔN isoforms by activating P2 promoters. The induced ΔN isoforms, in turn, inhibit TA isoforms. A good example of these interactions is an induction of Δ133p53 by p53 [5
]. Similarly, TAp73 and p53 are important regulators of transcriptions of ΔNp73 [39
]. It appears that the balance between ΔN and TA isoforms is finely tuned to regulate the activities of TA isoforms. The net effect of these interactions in a given context appears to be dependent on the TA/ΔN expression ratio. Deregulation of this mechanism may lead to tumor development [40
]. However, it has become clear that the role of ΔN isoforms is multifaceted. The dominant negative concept cannot explain the complexity of all the interactions attributed to ΔN isoforms. Several studies reported that ΔN isoforms can retain transcription activity through additional transactivation domains.