SHORT REPORT A novel p21 WAF1/CIP1 transcript is highly dependent on p53 for its basal expression in mouse tissues Andrei L Gartel* ,1 , Senthil K Radhakrishnan 1 , Michael S Serfas 2,3 , Young H Kwon 2,4 and Angela L Tyner* ,1,2 1 Department of Medicine, 840 S Wood St, University of Illinois at Chicago, Chicago, IL 60612, USA; 2 Department of Biochemistry and Molecular Genetics, 900 S Ashland Ave., University of Illinois at Chicago, Chicago, IL 60607, USA p21 WAF1/CIP1 is an important transcriptional target of p53 and it plays a critical role in growth arrest after DNA damage. Here, we report the identification of a novel alternate mouse p21 transcript that is conserved in evolution. It differs from the classical p21 WAF1/CIP1 transcript in the first exon, which is located at approxi- mately 2.8kb upstream of transcriptional start site of p21 WAF1/CIP1 and is sandwiched between two p53 binding sites. This novel p21 transcript is present in most mouse tissues with highest levels of expression in the spleen. In contrast to the classical p21 WAF1/CIP1 transcript, this new transcript is highly dependent on p53 for its basal expression, as evidenced by its absence in nearly all of p53 / mouse tissues. This transcript is also absent at nonpermissive temperature in a 10-1 mouse cell line lacking endogenous p53 and harboring temperature- sensitive p53 mutant. However, this novel transcript is induced to appreciable levels in the presence of high p53 activity at the permissive temperature. Our data suggest that p53-dependent induction of p21 may be an additive effect conferred by individual increases in the alternate and classical p21 transcripts. Oncogene (2004) 23, 8154–8157. doi:10.1038/sj.onc.1207820 Published online 13 September 2004 Keywords: p21; p53; alternate transcript p21 WAF1/CIP1 is the founding member of the Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) (reviewed in Gartel et al., 1996). It inhibits the activity of cyclin/Cdk2 complexes and plays an important role in cell cycle regulation (Brugarolas et al., 1999). p21 is one of the main downstream effectors of p53 function upon DNA damage (el-Deiry et al., 1993). Expression of p21 is controlled mostly at the transcriptional level by both p53-dependent and -independent mechanisms (reviewed in Gartel and Tyner, 1999). Overexpression of p21 arrests cells primarily in the G1phase of the cell cycle (Harper et al., 1995). Alternative splicing or alternate promoter usage can lead to the expression of different transcripts from the same locus (reviewed in Landry et al., 2003). These mechanisms have been shown to regulate a number of CKIs, including p15 INK4B (Tsubari et al., 1997), p16 INK4a- ARF (Duro et al., 1995; Mao et al., 1995; Quelle et al., 1995), p18 INK4c (Phelps et al., 1998), p57 KIP2 (Tokino et al., 1996) and human p21 WAF1/CIP1 (Nozell and Chen, 2002). Transcripts from a single locus may encode the same protein, as in p18 INK4c (Phelps et al., 1998), or they may encode different proteins as seen in p16 INK4a-ARF locus, where one transcript encodes INK4, which functions as a CKI, and the other encodes ARF, which is an inhibitor of MDM2 (Quelle et al., 1995). The human p21 WAF1/CIP1 locus produces three different transcripts utilizing two different promoters (Nozell and Chen, 2002). p21B and p21C are expressed from a promoter located within the first intron of human p21A (the classical human p21 WAF1/ CIP1 transcript; el-Deiry et al., 1993). While p21C encodes the normal p21 CKI, the p21B transcript produces a short protein of 123 amino acids that causes apoptosis when overexpressed. Upon DNA damage, p53 induces p21B and p21C transcripts through a newly identified p53 response element in their proximal promoter (Nozell and Chen, 2002). In this study, we have analysed the mouse p21 genomic locus and found that a novel transcript with a different first exon is expressed, and that its expression is highly dependent on p53. We discovered this alterna- tively spliced p21 transcript using the BLAST search tool against the mouse EST database. The first exon of this spliced form (hereafter called transcript-1) is approximately 2.8 kb upstream from the transcription start site of the classical p21 WAF1/CIP1 transcript (el-Deiry et al., 1993) (hereafter called transcript-2) and it is conserved in rat and human (Figure 1b). These two transcripts are shown in the genomic context in Figure 1a, along with one other transcript (hereafter Received 26 February 2004; revised 8 April 2004; accepted 16 April 2004; published online 13 September 2004 *Correspondence: AL Gartel; E-mail: agartel@uic.edu and AL Tyner, Department of Medicine, 840 S Wood St, University of Illinois at Chicago, Chicago, IL 60612, USA; E-mail: atyner@uic.edu 3 Current address: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI 53706, USA 4 Current address: Kookmin University, School of Techno-Science, Seoul, 136-702, Korea Oncogene (2004) 23, 8154–8157 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc