Differential Recognition of Response Elements Determines Target Gene Specificity forp53 and p63

Abstract

p63 is a member of the p53 tumor suppressor gene family, which regulates downstream target gene expression by binding to sequence-specific response elements similar to those of p53. By using oligonucleotide expression microarray analysis and analyzing the promoters of p63-induced genes, we have identified novel p63-specific response elements (p63-REs) in the promoter regions of EVPL and SMARCD3. These p63-REs exhibit characteristic differences from the canonical p53-RE (RRRCWWGYYY) in both the core-binding element (CWWG) as well as the RRR and/or YYY stretches. Luciferase assays on mutagenized promoter constructs followed by electromobility shift analysis showed that p53 preferentially activates and binds to the RRRCATGYYY sequence, whereas p63 preferentially activates RRRCGTGYYY. Whereas EVPL protein is highly expressed in epithelial cells of the skin and pharynx in the p63 ؉/؉ mouse, it is undetectable in these tissues in the p63 ؊/؊ mouse. Our results indicate that p63 can regulate expression of specific target genes such as those involved in skin, limb, and craniofacial development by preferentially activating distinct p63-specific response elements.p63 is a member of the p53 tumor suppressor gene family. Similar to p53, p63 is a transcription factor that activates target genes through sequence-specific DNA binding (35,41,43,52,56). It has been shown that expression of p21 waf-1 , MDM2, and BAX are induced by TAp63s through binding to p53 response elements (p53-REs) (45). In spite of their structural similarities, p63 functions differ greatly from those of p53. The most striking difference is the apparent involvement of p63 in skin and limb development. The p63 knockout mouse exhibits skin and limb defects as well as craniofacial abnormalities (29, 57). On the other hand, the p53 knockout mouse develops normally but is prone to suffering from various cancers from an early age (7). Heterozygous p63 germ line mutations cause several skin and other developmental disorders (1,3,17,28,53). On the other hand, germ line mutations of p53 cause Li-Fraumeni syndrome, in which affected individuals are exceptionally prone to developing cancer (26). p63 complements p53-dependent apoptosis induced by DNA damage. However, p63 itself induces apoptosis to a lesser extent than p53 (12, 42).These differences may be due to the differential regulation of target genes by p53 and p63. The p53 and p63 proteins can bind to two or more tandem repeats of RRRCWWGYYY (p53-RE) or some other motifs and subsequently activate target gene expression (5, 9, 54, 56). In the case of the 14-3-3 promoter, p53 and p63 differentially bind to two distinct response elements (55). Until now, a number of genes have been reported to be targets of p63 and its close relative, p73, such as JAG1, JAG2, IL4R, ⌬Np73, AQP3, and REDD1 (11,30,39,40,59). However, p63-specific response elements (p63-REs) have not yet been defined. Thus, the specific mechanism of gene activation exhibited by p63 and its distinction from that exhibited by p53 remain unclear.In order...

p63 consensus DNA-binding site: identification, analysis and application into a p63MH algorithm

“…Our data support the Osada et al (2005) findings and extend the characterization of the p63 DBS by defining the sequence-specific Representative results of replicate experiments are shown. Baseline, untreated lysates; ADR, adriamycin treatment for 6 h; pos., genomic DNA template; neg., no DNA template; and IP, immunoprecipitation.…”

“…Representative results are displayed as relative copy number of genomic fragments containing p21 RE1, PTPN14 RE and ING1 RE. p63 consensus DNA-binding site CA Perez et al characteristics of the tenth and eleventh position, which was not apparent from the work of Osada et al (2005). However, upon analysis of their published data, it is evident that the greatest difference in p63 versus p53 transactivation occurred with sequences containing a 5G, 10G and 11C combination.…”

“…Osada et al (2005) reported that p63 has higher specificity for CGTG at positions 4 to 7 in the 10 bp half sites of the 20 bp motif, especially in the setting of multiple mismatches from the p53 consensus RE. In contrast, Ortt and Sinha (2006) concluded that the optimal p63 DNA-binding motif contains a half-site with central core sequence CATG similar to p53's, but differs by having AT-rich 5 0 and 3 0 sequences flanking it.…”

“…The effect of an 11C change was sequence dependent, as only p63 had enhanced transcriptional activity on the 11CI RE, but both p63 and p53 had increased activity on the 11CII RE. The presence of a 10 bp motif with a CATG core in the 11CII RE, instead of the CAAG core in the 11CI RE, likely contributes to high p53 transactivation activity from the 11CII site (Inga et al, 2002;Osada et al, 2005). This could compensate for any detrimental effect of a deviation from the p53 canonical consensus at position 11.…”

p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress

“…Recent studies indicate that p63 proteins can bind DNA through response element (p63RE) slightly different to p53RE conferring responsiveness to p63 but not p53 proteins. 18,19 The DNp63 isoforms can bind DNA through p53RE and can exert dominant-negative effects over p53, p73 and p63 activities by either competing for DNA binding sites or by direct protein interaction (for a review Benard et al 20 ). DNp63 isoforms were also shown to directly activate specific gene targets not induced by TA isoforms.…”