Molecular Cell, Vol. 8, 57–69, July, 2001, Copyright 2001 by Cell PressTranscriptional Regulation by p53 throughIntrinsic DNA/Chromatin Bindingand Site-Directed Cofactor Recruitmentinhibitory C-terminal domain (290–325 and 356–393)(Hupp et al., 1992). This inhibition can be relieved byacetylation, phosphorylation, or protease cleavage.After DNA damage, p53 is phosphorylated and acet-ylated at a number of sites within its N- and C-termini.Joaquin M. Espinosa and Beverly M. Emerson1Regulatory Biology LaboratoryThe Salk Institute for Biological Studies10010 North Torrey Pines RoadLa Jolla, California 92037Phosphorylation within the N-terminal activation domainmost likely affects its interactions with Mdm2, whichcontrols p53 stability, and components of the transcrip-Summarytion initiation machinery (Prives, 1998). Acetylation ofp53 by the histone acetyl transferases CBP/p300 andThe tumor suppressor protein, p53, plays a critical rolePCAF activates DNA binding in vitro and each HAT canin mediating cellular response to stress signals bycoactivate p53-dependent transcription in transient ex-regulating genes involved in cell cycle arrest and apo-pression experiments (Gu and Roeder, 1997; Avantaggi-ptosis. p53 is believed to be inactive for DNA bindingati et al., 1997; Lill et al., 1997; Scolnick et al., 1997; Liuunless its C terminus is modified or structurally al-et al., 1999).tered. We show that unmodified p53 actively binds toAlthough a wealth of information exists concerningtwo sites at ⫺1.4 and ⫺2.3 kb within the chromatin-p53, little is known about the actual mechanism by whichassembled p21 promoter and requires the C terminusthis critical tumor suppressor protein directly interactsand the histone acetyltransferase, p300, for transcrip-with its target genes and regulates their expression. Wetion. Acetylation of the C terminus by p300 is not nec-have begun to address these issues by developing aessary for binding or promoter activation. Instead,p53-dependent in vitro transcription system using chro-p300 acetylates p53-bound nucleosomes in the p21matin-assembled p21 promoter-driven genes. The p21promoter with spreading to the TATA box. Thus, p53gene is a natural and direct target of p53 and is regulatedis an active DNA and chromatin binding protein thatin an inducible (El-Deiry et al., 1993) and a constitutivemay selectively regulate its target genes by recruit-basal level manner (Tang et al., 1998) through two con-ment of specific cofactors to structurally distinct bind-sensus p53 promoter binding sites at ⫺2.3 kb (5⬘) anding sites.⫺1.4 kb (3⬘). The p53 activation domain interacts withCBP/p300, and both CBP/p300 and PCAF increase theIntroductionability of p53 to activate p21 gene expression in vivo(Scolnick et al., 1997).The p53 tumor suppressor gene is the most frequentUsing the natural p21 promoter, we show that p53target for genetic alterations in cancer, with mutationsfunctions synergistically with p300 to activate transcrip-occurring in approximately 50% of all human tumors.tion through chromatin from a distance of at least 1.4The importance of p53 in cancer prevention results fromkb. Interestingly, p300 mediates p53-dependent tran-its ability to regulate such critical processes as cell cyclescription without increasing p53 binding to the chroma-progression and apoptosis (recent reviews Levine, 1997;tin template. Instead, p53 associates with its nucleoso-Prives and Hall, 1999; Vogelstein et al., 2000). p53 is amal target sites in the absence of chromatin remodelingsequence-specific DNA binding protein that has beenor modifying complexes. Chromatin-bound p53 then re-shown to activate transcription of a number of targetcruits p300 to the p21 promoter, resulting in localizedgenes, including p21, Mdm2, GADD45, Bax, and cyclinnucleosome acetylation with regional spreading to theG. Induction of p21 results in cell cycle arrest in responseTATA box. p300 mediates p21 gene expression by p53-to DNA damage by inhibiting cyclin-dependent kinasetargeted nucleosomal acetylation rather than throughactivity (El-Deiry et al., 1993; Xiong et al., 1993). Activa-p53 acetylation, which does not affect its transcriptionaltion of Bax and other genes by p53 promotes apoptosisactivity in vitro.(Miyashita and Reed, 1995). The regulation of cell cycleIn contrast with current views, we find that p53 is notarrest and apoptosis by p53 are mechanistically distincta latent but an active DNA binding protein that doesprocesses (Attardi et al., 1996). Recently, oligonucleo-not require modification of the C-terminal domain bytide microarray analysis has identified a broad spectrumacetylation or antibody binding to interact with eitherof genes that are controlled by p53 in a positive orDNA or chromatin. Our studies indicate that the C termi-negative manner, and whose functions fall into catego-nus is not inhibitory for p53 binding and is, in fact, re-ries that reflect the role of p53 as an integrator of diversequired for p53 association with particular target sites.cellular signals (Zhao et al., 2000).p53 interacts with distinct types of nucleosomal bindingThe 393 amino acid p53 protein consists of two N-ter-sites within the p21 promoter, as shown by differentminal activation domains (amino acids 1–42; 42–83), abehaviors in response to C terminus perturbations. Insequence-specific DNA binding domain (102–292), andaddition, p53 associates with chromatin at higher affinitya C-terminal oligomerization domain (324–355). The DNAthan with DNA in the absence of cofactors or proteinbinding activity of p53 is thought to be under negativemodifications. Thus, transcriptional regulation by p53constitutive regulation through two regions within itsmay be a complex property of chromatin structure, DNAtopology, and recruitment of specific cofactors to allo-sterically-regulated binding sites.1Correspondence: [email protected] Cell58Figure 1. Full-Length p300 Efficiently Acet-ylates p53(A) SDS-polyacrylamide gel analysis of re-combinant p53 and p300. Epitope-tagged hu-man full-length p53 and p300 were expressedin baculovirus-infected cells and affinity pu-rified.(B) Western blot of recombinant and partiallypurified native p300 from HeLa cells.(C) Acetyl transferase activity of recombinantp300. p300 was incubated with p53 or freecore histones in the presence of3H-acetylCoA. Reactions were electrophoresed andvisualized by Coomassie staining (left) or fluo-rography to detect acetylated proteins