Global transcriptional program of p53 target genes during the process of apoptosis and cell cycle progression Asra Mirza 1,6 , Qun Wu 1,6 , Luquan Wang 2 , Terri McClanahan 3 , W Robert Bishop 1 , Ferdous Gheyas 4 ,WeiDing 2 , Beth Hutchins 5 , Tish Hockenberry 1 , Paul Kirschmeier 1 , Jonathan R Greene 2 and Suxing Liu n,1 1 Tumor Biology Department, Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-4 (4600), Kenilworth, NJ 07033, USA; 2 Human Genomic Research Department, Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-4 (4600), Kenilworth, NJ 07033, USA; 3 DNAX Research Institute, 901 California Ave. Palo Alto, CA 94304, USA; 4 Biostatistics Group, Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-4 (4600), Kenilworth, NJ 07033, USA; 5 Canji Inc., 3525 Johns Hopkins Court, San Diego, CA 92121, USA The temporal gene expression profile during the entire process of apoptosis and cell cycle progression in response to p53 in human ovarian cancer cells was explored with cDNA microarrays representing 33 615 individual human genes. A total of 1501 genes (4.4%) were found to respond to p53 (approximately 80% of these were repressed by p53) using 2.5-fold change as a cutoff. It was anticipated that most of p53 responsive genes resulted from the secondary effect of p53 expression at late stage of apoptosis. To delineate potential p53 direct and indirect target genes during the process of apoptosis and cell cycle progression, microarray data were combined with global p53 DNA-binding site analysis. Here we showed that 361 out of 1501 p53 responsive genes contained p53 consensus DNA-binding sequence(s) in their regulatory region, approximately 80% of which were repressed by p53. This is the first time that a large number of p53-repressed genes have been identified to contain p53 consensus DNA- binding sequence(s) in their regulatory region. Hierarch- ical cluster analysis of these genes revealed distinct temporal expression patterns of transcriptional activation and repression by p53. More genes were activated at early time points, while more repressed genes were found after the onset of apoptosis. A small-scale quantitative chro- matin immunoprecipitation analysis indicated that in vivo p53–DNA interaction was detected in eight out of 10 genes, most of which were repressed by p53 at the early onset of apoptosis, suggesting that a portion of p53 target genes in the human genome could be negatively regulated by p53 via sequence-specific DNA binding. The ap- proaches and genes described here should aid the under- standing of global gene regulatory network of p53. Oncogene (2003) 22, 3645–3654.doi:10.1038/sj.onc.1206477 Keywords: p53; transcription; microarray; chromatin immunoprecipitation; bioinformatics Introduction p53 is a central regulator of cell growth, DNA damage repair, and apoptosis (Vogelstein and Levine, 2000). It is a transcription factor that specifically recognizes DNA sequences containing two adjacent copies of the consensus sequence 5 0 -RRRCWWGYYY(N ¼ 0– 13)RRRCWWGYYY-3 0 (El-Deiry et al., 1992). In this motif, R ¼ G or A, W ¼ T or A, Y ¼ C or T, N ¼ any base. p53 directly activates the expression of a sub- stantial number of genes important for cell cycle regulation and apoptosis, through binding to the p53 DNA-binding site(s) (El-Deiry et al., 1993; Hollander et al., 1993; Buckbinder et al., 1995; Miyashita and Reed, 1995; Ludes-Meyers et al., 1996; Morris et al., 1996). Identification of transcriptional targets of p53 is of great importance in understanding the pathways by whichp53regulatesgrowtharrestandapoptosis.Recent studies using microarray or SAGE analysis aimed at identifying differentially expressed genes in response to p53 have yielded valuable insights into these mechan- isms (Madden et al., 1997; Polyak et al., 1997; Yu et al., 1999; Maxwell and Davis, 2000; Zhao et al., 2000). Although numerous genes have been identified, a mixed readout of direct and indirect p53 target genes is obtained from these experiments. In a recent study, we reported on a p53 Target Database, consisting of 4852 human genes that contain at least one p53 consensus binding sequence in their regulatory region (Wang et al., 2001). Availability of this database provides an im- portant tool for identifying p53 target genes at the genomic scale. In contrast to transcriptional activation, most genes reported to be repressed by p53 lack a classical p53- binding site in their promoter (Gopalkrishnan et al., 1998; Kaluzova et al., 2000; Krause et al., 2000). Several mechanisms have been proposed for p53-mediated repression, including the sequestration of components of the basal transcription machinery by p53 (Liu et al., 1993; Lu and Levine, 1995; Gopalkrishnan et al., 1998), regulation by histone deacetylases (Murphy et al., 1999; Received 2 December 2002; revised 5 February 2003; accepted 12 February 2003 *Correspondence: S Liu; E-mail: suxing.liu@spcorp.com. 6 These authors equally contributed to this work Oncogene (2003) 22, 3645–3654 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc ONCOGENOMICS