Molecular Cell, Vol. 8, 57–69, July, 2001, Copyright 2001 by Cell Press

Transcriptional Regulation by p53 throughIntrinsic DNA/Chromatin Bindingand Site-Directed Cofactor Recruitment

inhibitory 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. Emerson1

Regulatory Biology LaboratoryThe Salk Institute for Biological Studies10010 North Torrey Pines RoadLa Jolla, California 92037

Phosphorylation 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 to

Although 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 frequent

Using 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 the

G. 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 through

activity (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 apoptosis activity in vitro.(Miyashita and Reed, 1995). The regulation of cell cycle In contrast with current views, we find that p53 is notarrest and apoptosis by p53 are mechanistically distinct a 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 spectrum acetylation or antibody binding to interact with eitherof genes that are controlled by p53 in a positive or DNA 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 diverse quired for p53 association with particular target sites.cellular signals (Zhao et al., 2000). p53 interacts with distinct types of nucleosomal binding

The 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), a behaviors in response to C terminus perturbations. Insequence-specific DNA binding domain (102–292), and addition, p53 associates with chromatin at higher affinitya C-terminal oligomerization domain (324–355). The DNA than with DNA in the absence of cofactors or proteinbinding activity of p53 is thought to be under negative modifications. Thus, transcriptional regulation by p53constitutive regulation through two regions within its may be a complex property of chromatin structure, DNA

topology, and recruitment of specific cofactors to allo-sterically-regulated binding sites.1 Correspondence: emerson@salk.edu

Molecular Cell58

Figure 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 of 3H-acetylCoA. Reactions were electrophoresed andvisualized by Coomassie staining (left) or fluo-rography to detect acetylated proteins (right).(D) p53 acetylation by p300 activates p53DNA binding activity. Equal amounts of nativeor acetylated p53 were incubated with a 32P-labeled 25 bp oligonucleotide containing aconsensus p53 binding site (p21 promoter, 5�

site) and analyzed by EMSA.

Results insect cells is mainly nonacetylated, an observation thatis further supported by Western blot assays using anti-bodies that specifically recognize the acetylated formSynergistic Transcriptional Activation

of the Chromatin-Assembled p21 of p53 (see Figure 3C). This experiment also suggeststhat other modifications previously reported to activatePromoter by p53 and p300

We expressed full-length human p53 and full-length hu- p53 DNA binding activity are absent in these prepara-tions.man p300 in insect cells using the baculovirus expres-

sion system. Flag-tagged p53 and His-tagged p300 were To analyze the mechanism of p53-dependent tran-scriptional activation of chromatin-assembled p21 pro-purified by affinity chromatography to apparent homo-

geneity (Figure 1A). The full-length size of recombinant moters, we used a DNA construct containing a 2.4 kbfragment of the human p21 promoter. This includes twop300 was confirmed by comparative Western blot, using

native p300 as control (Figure 1B). When p300 acetyl- binding sites for p53, which are centered around posi-tions �2270 (5� site) and �1380 (3� site), respectivelytransferase activity was assayed using core histones

and p53 as substrates, p300 efficiently acetylated p53 (Figure 2A). This promoter fragment drives the transcrip-tion of a luciferase reporter gene (WWP-LUC or p21-and histones H3 and H4 in solution and histones H2A and

H2B to a lesser extent (Figure 1C). This experiment LUC). This 8 kb plasmid has been extensively used tostudy p53-dependent transcription of the p21 promotershows that full-length p300 can acetylate p53, as pre-

viously reported for its histone acetyl-transferase (HAT) by transient expression experiments, and the require-ment of at least one of these two binding sites for p53-domain alone (Gu and Roeder, 1997).

The p53 protein obtained from insect cells was mainly dependent transactivation has been amply demon-strated (El-Deiry et al., 1995).inactive for specific DNA binding activity when mea-

sured by EMSA using a 25 bp oligonucleotide harboring Chromatin assembly of this template DNA was per-formed using Drosophila embryo cell-free extracts andthe 5� site in the p21 promoter. In agreement with previ-

ous experiments using only the catalytic domain of p300, purified core histones as previously described (Bulgerand Kadonaga, 1994). The quality of the reconstitutedthe DNA binding activity of p53 was dramatically im-

proved by acetylation using full-length p300 (Figure 1D). chromatin was assessed by transcriptional repressionand structural analysis by micrococcal nuclease diges-The efficiency of activation by p300-mediated acetyla-

tion was comparable to that observed with other known tion (Figure 2C, lanes 5–6, and Figure 2D). The micrococ-cal nuclease digestion pattern of the in vitro reconstitu-activating stimuli, such as binding of the antibody

PAb421 to the C-terminal region of the protein or dele- ted chromatin showed the presence of physiologicallyspaced nucleosomes and was correlated with a com-tion of the C terminus itself (see Figures 4B and 5B). Our

analyses demonstrate that the p53 protein produced in plete repression of p21 promoter activity.

Transcriptional Regulation of Chromatin by p5359

Figure 2. p53 and p300 Can Direct Synergistic Transcriptional Activation from Chromatin-Assembled p21 Promoters

(A) Schematic diagram of the p21 promoter. Relative positions to the transcription initiation site and sequences of the p53 binding sites areindicated.(B) Schematic representation of the in vitro chromatin assembly and transcription protocol.(C) p53-dependent transcription of chromatin-assembled p21 promoters by addition of p53 � p300. �-globin is shown as an internal control.(D) Analysis of p21-LUC chromatin structure by micrococcal nuclease digestion.

The p21-LUC chromatin template was subjected to line, erythroid K562 (data not shown). Thus, transcrip-tional activation from the chromatin-assembled p21 pro-in vitro transcription reactions using HeLa cell nuclear

extracts as a source of basal initiation components in the moter in vitro requires p53, acting at a distance of at least1.38 kb, in combination with a histone acetyl transferasepresence or absence of p53 and/or p300, as described in

the protocol depicted in Figure 2B. Both factors were cofactor, p300. Interestingly, other putative cofactors,such as PCAF and human SWI/SNF, do not facilitateadded after chromatin assembly was complete (“post

assembly”) and incubated with the template prior to in p53-dependent transcription in these assays eventhough they both interact with p53 (our unpublishedvitro transcription. Addition of p53 alone produced very

modest transcriptional activation (Figure 2C, lane 7). data).However, coincubation with p300 gave a strong syner-gistic de-repression of the chromatin template (lane 8). p300 Can Mediate p53-Dependent Transcription

without Increasing p53 Bindingp300 alone was unable to achieve such activation (lane9). Neither p53 nor p300 appreciably influenced tran- to the Chromatin Template

To analyze the mechanism by which p300 mediates p53-scription of naked DNA templates (lanes 1–4). Identicalresults were obtained using extracts from another cell dependent activation, we performed DNase I chromatin

Molecular Cell60

Figure 3. p300 Mediates p53-Dependent Transcription without Enhancing Its Interaction with Chromatin

After incubation of chromatin with p53 � p300, the sample was split into three aliquots and tested in separate assays:(A) DNase I chromatin footprinting of the 5� and 3� p53 binding sites in the p21 promoter. Bars at the left indicate the position of thecorresponding binding sites. Bars at the right indicate regions of protection (�) or hypersensitivity (�) to DNase I digestion.(B) In vitro transcription of p21 chromatin with p53 � p300.(C) Western blot analysis of chromatin samples containing p53 � p300. Membranes were probed with antibodies against anti-acetyl p53 (top)or p53 N-terminal domain (bottom).

footprinting assays to examine the occupancy of the 5� These observations raised some interesting questionsabout the coactivation mechanism of p300 and the DNA/and 3� sites of the p21 promoter under conditions of

transcriptional coactivation. Chromatin assembly and chromatin binding properties of p53. For example, isunmodified p53 able to interact with its chromatin bind-factor incubation steps were performed as described

for Figure 2 but the samples were split into three aliquots ing sites as well as the acetylated isoform in contrastto what is observed in EMSA for short pieces of nakedjust before the in vitro transcription reaction. One aliquot

was subjected to DNase I digestion, the second was DNA? Can p53 interact with its chromatin binding siteson its own or does it require chromatin modifying ortranscribed, and the third was analyzed by Western blot

to determine the acetylation status of p53. Surprisingly, remodeling activities present in the assembly extract?p53 alone was able to generate a clear footprinting pat-tern at both the 5� and 3� binding sites within the p21 p53 Does Not Require Acetylation or Remodeling

Complexes to Interact with Chromatinpromoter (Figure 3A, lanes 2 and 6). Addition of p300did not significantly change the footprinting pattern pro- To answer these questions, we performed footprinting

experiments with chromatin-assembled p21 promotersduced by p53, even though it clearly enhanced tran-scription from the p21 promoter (Figure 3B, lane 4). In after ATP depletion and purification by gel filtration using

nonsaturating amounts of p53. These chromatin sam-a parallel experiment using the same proteins and re-agents, p300 clearly activated p53 DNA binding activity ples are devoid of endogenous ATP-dependent nucleo-

some remodeling activities, small molecules like ATPin EMSA (data not shown, similar to Figure 1D).The acetylation status of both untreated and pre- or acetyl CoA, and the majority of proteins from the

chromatin assembly extract (Mizuguchi and Wu, 1999).viously acetylated p53 was preserved during incubationwith the chromatin template as determined by Western As shown in Figure 4A, both native and previously acet-

ylated p53 are able to interact with the 5� and 3� bindingblot analyses using antibodies that specifically recog-nize only the acetylated form of p53 (Figure 3C). These sites in the purified p21 promoter chromatin with similar

affinities.antibodies did not recognize native p53, whereas thep300-acetylated p53 isoform gave a strong signal. Thus, From this experiment, we draw three main conclu-

sions. First, although native p53 possesses an almostnative p53 remained unacetylated, and previously acet-ylated p53 was not deacetylated during the chromatin undetectable DNA binding activity when assayed on

short oligonucleotides, it binds very efficiently to theassembly incubation. We conclude from this experimentthat p300 can mediate p53 transactivation without af- same sites in a larger promoter context when assembled

into chromatin. Second, whereas acetylation of p53 byfecting the ability of p53 to bind to its DNA recognitionsequences in chromatin. p300 dramatically increases its affinity for short DNA

Transcriptional Regulation of Chromatin by p5361

Figure 4. Interaction of p53 with Its Chromatin Binding Sites in the p21 Promoter Is Not Enhanced by Acetylation or by Antibodies Directedagainst the C-Terminal Domain

Chromatin reactions were depleted of ATP by treatment with apyrase and further purified by gel filtration prior to DNaseI footprinting.(A) Chromatin templates were incubated with increasing subsaturating concentrations of untreated or previously acetylated p53 (between 1and 20 nM) and processed as described.(B) EMSA analysis of different p53 isoforms. The binding of p53 to a 25 bp oligonucleotide (5� site) was tested � the antibody PAb421. Acontrol with p300-acetylated p53 was also included.(C) Binding of PAb421 antibody to the C-terminal domain of p53 inhibits its ability to occupy the 3� p21 promoter chromatin binding site.

Molecular Cell62

duplexes, it does not increase its affinity for the same insight into this issue, we examined a C-terminal deletedform of p53, lacking the last 30 amino acid residues (p53sequences in p21 promoter chromatin. Third, native p53�C30). After production in insect cells and comparativeinteracts efficiently with its nucleosomal binding sitesquantification with wild-type p53 (Figure 5A), we testedin the absence of chromatin modifying or remodelingthe binding activity of this protein in both EMSA andactivities.DNase I footprinting. As expected according to previousreports, the �C30 mutant was strongly active in thep53 Possesses Distinct Types of ChromatinEMSA analysis as compared with the acetylated formand DNA Binding Sitesof wild-type p53 (Figure 5B). By contrast, when the �C30To gain more insight into the interaction of p53 withmutant was assayed by DNase I footprinting on p21chromatin, we made use of a different tool commonlypromoter DNA or chromatin, it showed a weaker bindingemployed to activate its dormant DNA binding activity.to the 5� site and no binding to the 3� site (Figure 5C).Previous reports have concluded that the C-terminalWe conclude that the C-terminal domain is not inhibitorydomain of p53 inhibits its ability to interact with DNA.for DNA binding and that it is required for interactionSuch inhibition can be artificially relieved by binding awith both the 5� and 3� p21 promoter sites (to differentmonoclonal antibody to the C-terminal domain (Huppextents) when these are present in larger molecules ofet al., 1992). Interestingly, the binding of the monoclonalDNA or chromatin. This is consistent with a functionalantibody PAb241 does not increase the affinity of p53analysis showing that the C-terminal domain is requiredfor all DNA binding sites tested. Moreover, the affinity forfor in vitro transcription from the chromatin-assembledsome binding sites is significantly decreased, whereas itp21 promoter (Figure 5D).remains unchanged for others. Similar behavior has

The failure to detect latent DNA binding activity ofbeen observed for other “activating” modifications, sug-p53 that can be activated by acetylation, incubationgesting the existence of different types of DNA bindingwith the PAb421 antibody, or deletion of the C-terminalsites for p53. In this regard, the two p53 recognitiondomain raises an important issue. Such latency is clearlysequences in the p21 promoter fall into different catego-evident when measuring p53 DNA binding activity byries: binding to the 5� site is strongly enhanced by treat-EMSA using short duplexes of naked DNA, but it is notment with PAb421, whereas binding to the 3� site isapparent when the same binding sites exist within ainhibited when measured by EMSA (Resnick-Silvermanlarger DNA context with or without chromatin assembly.et al., 1998). Therefore, we decided to test the effect ofThe resolution of this discrepancy is critical toward un-PAb421 on p53 binding to these two binding sites whenderstanding the regulation of p53 DNA binding activity.assembled into chromatin.

As shown in Figure 4B, the addition of PAb421 toThe Affinity of p53 for the 5� p21 Promoter Bindingnative p53 increases its DNA binding activity for theSite Increases as a Function of Target DNA Size5� site to a similar extent as that observed by p300-There are many differences between the two DNA bind-dependent acetylation when examined by EMSA. Asing assays performed in this study, EMSA and chromatinexpected, the protein-DNA complex was supershiftedor DNA plasmid footprinting. First, the size of the tem-by the presence of the antibody. However, when exam-plate DNA is much longer in the footprinting experimentsined by chromatin footprinting (Figure 4C), treatment(25 bp for EMSA versus 8 kb for footprinting). Second,with PAb421 did not increase the affinity of p53 for theprotein-DNA binding occurs in solution in footprinting5� site, similar to our previous observations with acetyla-reactions, whereas binding continues inside the poly-tion by p300 (Figure 4A). Interestingly, PAb421 clearlyacrylamide gel in EMSA, which can affect the formation

blocked the binding of p53 to the 3� site (Figure 4Cor stability of the complex.

bottom). Thus, using chromatin-assembled p21 promot-To discriminate between these alternatives, we per-

ers, the antibody retained its inhibitory effect on p53 formed EMSA with native and acetylated p53 usinginteraction with the 3� site, but it lacked the stimulatory pieces of double-stranded naked DNA of increasingeffect on the 5� site, in agreement with previous reports size. Probes were generated that contained a centered(Kim et al., 1997; Cain et al., 2000). 5� site of the p21 promoter with increasing flanking re-

This chromatin footprinting analysis allows us to make gions from the same promoter which are devoid of anytwo major conclusions. First, two different putative acti- other p53 binding sites. A typical result is shown invating modifications of native p53, namely acetylation Figure 6A in which binding of p53 to a 25 bp oligonucleo-and binding of the antibody PAb421, fail to improve the tide was increased 12-fold by acetylation, whereas bind-ability of p53 to interact with the chromatin-assembled ing to a 160 bp DNA molecule was increased only 1.5-p21 promoter. Second, different types of p53 binding fold. This decrease in the influence of acetylation wassites exist within this promoter as revealed by the selec- due to the fact that unacetylated p53 had increasedtive inhibition of binding to only the 3� site by PAb421. affinity for the longer piece of DNA. The presence of

additional p53 binding sites in the longer molecule isThe C-Terminal Domain of p53 Is Not Inhibitory unlikely because we detected only one shifted complexand Is Differentially Required for Binding in EMSA and only one protected site within this regionto Distinct DNA Sequences in a parallel footprinting experiment (Figure 6B). The lackThe observation that two modifications of the C-terminal of effect of acetylation on p53 binding is most apparentdomain, acetylation and binding of the antibody PAb421, when analyzed on the p21 promoter within an 8 kb plas-do not enhance the interaction of p53 with the p21 pro- mid. In this case, native and acetylated isoforms of p53moter argues against the idea that the C-terminal do- showed indistinguishable affinities for the 5� binding site

as determined by DNase I footprinting (Figure 6B).main inhibits the DNA binding activity of p53. To gain

Transcriptional Regulation of Chromatin by p5363

Figure 5. The C-Terminal Domain of p53 Is Not Inhibitory and Is Differentially Required for Binding to Distinct DNA Sites

(A) Protein purification of recombinant wild-type (WT) and C-terminal deletion mutant of p53 (�C30).(B) EMSA analysis of �C30 mutant.(C) DNase I footprinting comparing the binding of wild-type p53 and �C30 to both the 5� site (top) and 3� site (bottom) of the p21 promoteras naked DNA (left column) or chromatin (right column).(D) In vitro transcription of p21 chromatin comparing wild-type p53 versus �C30 � p300.

These experiments indicate that p53 has very low plates, most significantly at the 5� site (Figure 6C, lanes2–10, compare top and bottom). Binding conditionsaffinity for its recognition sequences when present in

the form of short oligonucleotides, and that the binding were exactly the same for both DNA and chromatintemplates, and protein concentrations were equalizedimproves significantly when the flanking regions are ex-

tended. This type of behavior is very suggestive of the with purified BSA to avoid “protein carrier” effects. DNAconcentration was also the same in all reactions as as-presence of secondary structures in the binding sites

(Kim et al., 1997). Such structures cannot be adopted sessed by electrophoresis in agarose gels. BecauseDNA supercoiling strongly influences p53 binding withinby short oligonucleotides, but are firmly stabilized by

the addition of adjacent duplex regions. An alternative the MDM2 promoter (Kim et al., 1999), we examinedwhether the relaxation introduced by nucleosome as-explanation is the possible requirement of DNA bending

at the p53 binding sites for high affinity protein interac- sembly could explain the difference in the affinity fornaked DNA compared to chromatin. However, after re-tion (Nagaich et al., 1999). Interestingly, as the affinity

of p53 increases for DNA of a particular length and laxing supercoiled p21-LUC plasmids by treatment withtopoisomerase I, no differences in p53 binding to the 5�topology, it becomes indistinguishable from the acet-

ylated form. or 3� site were observed between relaxed and su-percoiled templates (data not shown).

Quantification of the occupancy of both 5� and 3� p21p53 Binds with Higher Affinity to Chromatinthan to DNA promoter sites was performed by densitometric analysis

of bands marked by asterisks (Figure 6C). This demon-We next assessed the relative binding affinities of p53for its two sites within the p21 promoter as DNA or strated a clear difference in the kinetics of p53 binding

to DNA or chromatin templates at both sites. Whereaschromatin. The interaction of some DNA binding pro-teins is impeded by nucleosomes, and specific remodel- p53 interaction with chromatin increased almost loga-

rithmically linear with protein concentration, binding toing complexes are required to facilitate their association(Armstrong et al., 1998; Kadam et al., 2000). To address DNA showed a clear “threshold” effect with a dramatic

change in occupancy within a narrow window of proteinthis issue for p53, we performed extensive protein titra-tions in parallel experiments using DNA or CL4B-purified concentration. This difference was more apparent for

the 3� site (lanes 13–21). Taken together, these datachromatin. Surprisingly, these experiments revealedthat p53 binds with higher affinity to nucleosomal tem- suggest that the structure of the two p21 promoter bind-

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Figure 6. p53 Sequence-Specific DNA Binding Affinity and Kinetics Are Sensitive to Target DNA Size and Chromatin Assembly

(A) EMSA analysis of p53 binding using DNA probes of different size. 25 and 160 bp oligonucleotides containing one centered copy of thep21 promoter 5� site were incubated with untreated or acetylated p53.(B) DNase I footprinting of p53 on naked DNA. Supercoiled p21-LUC plasmids were incubated with increasing concentrations of untreatedor acetylated p53 and processed as described.(C) p21-LUC plasmid DNA (top row) or CL4B-purified chromatin (bottom row) were used as templates for extensive titrations of wild-type p53on both the 5� and 3� sites of the p21 promoter. Binding conditions were identical for both templates, and the concentration of DNA in bothtypes of samples was equivalent.

ing sites may be altered within nucleosome arrays to promoters; and second, transactivation of the transcrip-generate a higher affinity for p53 and to influence its tional machinery. After observing that p300 is requiredassociation kinetics. This regulatory feature of chroma- to mediate p53-dependent activation of chromatin-tin could be of critical importance within cells when assembled p21 promoters (Figure 2) but does not facili-distinct p53 target genes are activated in response to tate the binding of p53 (Figures 3 and 4), we investigatedchanging levels of p53 protein. whether acetylation of p53 played an important role at

another step in the transcription process. The acetyla-tion pattern of p53 has been previously determined, andAcetylation of the C-Terminal Domain of p53 Doesmutations of lysine residues that are targeted by p300Not Influence Its Transcriptional Activityhave been generated and analyzed elsewhere (Gu andGene regulation by p53 occurs in discrete stages: first,

interaction with its binding sites in chromatinized target Roeder, 1997; Avantaggiati et al., 1997; Scolnick et al.,

Transcriptional Regulation of Chromatin by p5365

1997; Luo et al., 2000). We examined a mutant versionof p53 in which lysines 370, 372, 373, 381, and 382 withinthe C-terminal domain have been replaced by arginine(p53KR, Luo et al., 2000). p53KR was expressed in insectcells, purified, and analyzed for its ability to activatetranscription compared with wild-type p53. As shownin Figure 7A, the mutant protein is still coactivated byp300 to a similar extent as wild-type p53 (compare lanes4 and 6). This indicates that acetylation of the C-terminaldomain does not play a major role in either chromatinbinding or subsequent steps in the p300-mediated acti-vation process. A Western blot analysis confirms thatp53KR is not recognized by antibodies specific to acet-ylated p53 (Figure 7B). These results are in agreementwith in vivo transient expression experiments, whichdemonstrate that mutations in acetylatable lysines resultin only a slight or no decrease in p53-dependent trans-activation (Scolnick et al., 1997, Nakamura et al., 2000).

p53 Recruits p300 and Directs NucleosomeAcetylation of the p21 PromoterOur studies indicate that p300 does not coactivate p53function by either facilitating its interaction with chroma-tin or increasing its transactivation potential. We, there-fore, explored other mechanisms by which p300 maymediate p53-dependent transcription. Particularly whetherp53 recruits p300 to acetylate nucleosomal histoneswithin the p21 promoter. For this purpose, we assem-bled the p21 promoter into chromatin using defined fac-tors instead of Drosophila embryo extracts. This re-combinant chromatin assembly system, developed byKadonaga and colleagues, consists of the ACF complex(dACF � dISWI) and the histone chaperone NAP-1,which together catalyze the assembly of free histonesinto nucleosomes in the presence of ATP. This systemhas been used successfully to obtain regularly spacednucleosome arrays and allows a higher degree of manip-ulation compared to the embryo extracts (Ito et al.,1999). For our purposes, use of recombinant factorsenabled us to detect de novo acetylation of the chroma-tin-assembled p21 promoter by incorporation of exoge-nously added 3H-acetyl CoA. Under the optimal his-tone:DNA ratio, the majority of histones in the assemblyreaction are incorporated into a nucleosomal array. Thisis critical because it has been demonstrated recentlythat p300 alone can acetylate free histones but notnucleosomes unless recruited by the activator GAL4-VP16 (Ito et al., 2000).

The quality of p21 promoter chromatin assembled inFigure 7. The Role of p53 and Histone Acetylation in Transcriptional this purified system was determined by micrococcalCoactivation by p300 nuclease digestion, as for embryo extracts, with similar(A) The transcriptional activity of wild-type p53 was compared to results (data not shown). The templates were then incu-p53KR, in which five lysine residues of the C-terminal domain weremutated to arginine. Chromatin-assembled p21-LUC plasmids wereincubated with wild-type p53, mutant p53 KR, and p300 as indicated.(B) Western blot analysis of p53 acetylation. Prior to initiation of (D) Chromatin immunoprecipitation assays. Chromatin templatestranscription, small aliquots of the chromatin reactions were re- were incubated with p53 � p300 as in (C) and digested with micro-moved and assayed for stability of acetylated or unacetylated forms coccal nuclease prior to immunoprecipitation with antibodies toof p53. Antibodies against the N-terminal domain of p53 or the acetylated histone H4. Recovered DNA was employed as templateacetylated C-terminal domain of p53 were used. in PCR amplifications using primers against different regions of p21-(C) Chromatin HAT assay. p21 promoter plasmids assembled into LUC. All amplification products were approximately 150 bp longchromatin with purified components (dACF � NAP1) were incubated and contain the sequence elements indicated at the left of eachwith p53 � p300 in the presence of 3H-acetyl CoA. Acetylation reac- panel. The differences in precipitated DNA between lanes 9 and 11tions were analyzed by SDS-PAGE, and labeled proteins detected are indicated as fold-induction. Input DNA (5%) and minus antibodyby fluorography. controls were also included.

Molecular Cell66

bated with p53 and/or p300 in the presence of 3H-acetyl other components of the transcription machinery. p300-mediated transcriptional activation has been describedCoA and the extent of acetylation monitored by incorpo-for other chromatin-assembled genes (Kraus et al., 1999;ration of radioactivity when analyzed by SDS-PAGE andDilworth et al., 1999). Our experiments demonstrate thatfluorography (Figure 7C). When both p53 and p300 werea mechanism by which p300 can regulate the activitypresent in the reaction, a strong acetylation of all fourof natural promoters is by acetylating chromatin over anucleosomal histones was easily detected (lane 3),long-range when recruited by a distal transcription fac-whereas very weak acetylation was observed by p300tor. Targeted nucleosome acetylation by chromatin-alone (lane 4). A control reaction with equivalent amountsbound GAL4-VP16 has been described recently (Ito etof free histones and p300 was included (lane 5). Acetyla-al., 2000). Previous studies in yeast revealed a localizedtion of p53 and autoacetylation of p300 was also de-factor-directed acetylation encompassing 1–2 nucleo-tected (lanes 3–4). This experiment clearly demonstratessomes within the proximal promoters (Rundlett et al.,that p53 can recruit the HAT activity of its coactivator1998; Struhl et al., 1998). In the absence of p53, p300p300 to the chromatin template and mediate de novocannot acetylate nucleosomes due to lack of templateacetylation of nucleosomal histones.targeting, and the p21 promoter remains inactive. WeTo determine whether p53 influenced the localizationfind that p53 proteins containing mutations in lysineof nucleosome acetylation on the p21 promoter by directresidues acetylated by p300 are as active as wild-typerecruitment of p300, we performed in vitro chromatinp53 in regulating p21 transcription in vitro. This indicatesimmunoprecipitation assays. p21 promoters were as-that acetylation of p53 does not contribute to its trans-sembled into chromatin using purified components andactivation potential, and that p300 does not mediateincubated with p53 and/or p300. Chromatin was di-transcription by this mechanism in our biochemicalgested with micrococcal nuclease and acetylated oligo-assays. This conclusion is in agreement with previousnucleosomes were immunoprecipitated using anti-ace-in vivo analyses in which p53 mutants lacking thesetyl-H4 antibodies. Specific p21 plasmid sequences inlysine residues did not show a significant decrease inthe immunoprecipitates were detected by PCR amplifi-transcriptional activity (Scolnick et al., 1997, Nakamuracation (Figure 7D) or Southern slot blot (data not shown).et al., 2000). However, p53 acetylation may play a roleIdentical results were obtained from both assays. Asin protein stabilization or subnuclear localization (Naka-expected, the amount of DNA precipitated was greatlymura et al., 2000; Pearson et al., 2000).enhanced when both p53 and p300 were incubated with

Numerous studies have focused on the DNA bindingthe chromatin template (lane 11). The extent of increasedproperties of p53 and the role of distinct protein domainsacetylation in different regions of the p21-LUC plasmidin this process. This issue is especially germane be-varied from 3.4-fold near the 3� p53 binding site to 1.4-cause the majority of p53 mutations found in humanfold in a distal region located 2 kb downstream of thecancers occur within the DNA binding domain. Thus,proximal promoter. Interestingly, nucleosome acetyla-the inability of p53 to interact with its target genes andtion also occurs several hundred base pairs from the 3�regulate transcription would be expected to contributesite to reach the proximal promoter (TATA box). Wesignificantly to cancer development and progression.consistently obtained the following decreasing levels ofPrevious experiments have led to the conclusion thatacetylation: 3� site � TATA box � 5� site �� �2kb.p53 is a latent DNA binding protein which contains anThese data indicate that in the presence of p53, p300inhibitory C-terminal domain. Using primarily EMSAacetylates nucleosomes in a targeted manner withinanalyses, latent DNA binding of p53 has been shown tochromatin-assembled p21 promoter-containing plas-be activated in several ways: posttranslational modifica-mids (lane 11). In the absence of p53, only negligibletions of the C terminus, such as acetylation or phosphor-nucleosome acetylation by p300 is apparent (lane 12).ylation; association with the monoclonal antibodyInterestingly, acetylation is highest when p300 is re-PAb421; or deletion of the C-terminal domain (reviewed

cruited to the promoter proximal 3� p53 binding site andin Prives and Hall, 1999). In contrast to these observa-

then apparently spreads to encompass the TATA box.tions, our experiments demonstrate that unmodified,

Thus, the probable mechanism by which p300 coacti- full-length p53 binds very efficiently to its natural recog-vates p53-dependent transcription is by targeted nition sequences within both DNA and chromatin. Thisnucleosomal acetylation of the proximal promoter when occurs in the absence of p53 modification by acetylrecruited by bound p53 at a distance of at least 1.4 kb. transferases or chromatin disruption by ATP-dependent

remodeling complexes. In fact, perturbations of theDiscussion C-terminal domain reveal the distinct nature of individual

p53 binding sites. For example, deletion of the C termi-Our studies demonstrate that p53 activates transcription nus or association of this domain with PAb421 actuallyfrom a natural target promoter, p21, when bound at a abolishes p53 interaction with the 3� site within the p21distance of at least 1.4 kb in a chromatin environment. promoter. Conversely, both modifications result in theTranscriptional activation requires the histone acetyl- switch from latent to active DNA binding by p53 whentransferase, p300. Surprisingly, p300 does not function analyzed by EMSA. The discrepancy between p53 bind-by facilitating p53 binding to its DNA recognition sites ing results obtained with EMSA using short oligonucleo-within chromatin. Instead, p300 acts at a later step in tides and DNase footprinting using larger promoter frag-the transcription process by acetylating nucleosomes ments can be resolved by increasing the length of thewithin the proximal and distal p21 promoter when tar- oligonucleotides so that it presumably adopts a second-geted by bound p53. This presumably renders the ary structure to which p53 can stably bind. Importantly,

Cain et al. (2000) have shown that modifications of thenucleosomes sufficiently fluid to allow interaction with

Transcriptional Regulation of Chromatin by p5367

N terminus greatly affect p53 binding to the p21 pro- recruit cofactors may be determined by the conforma-moter (5� site) as measured in footprinting assays. This tion it assumes when bound to DNA/chromatin sitesis especially significant because the N terminus is the having specific topologies. Such allosteric regulation hassite of multiple posttranslational modifications and can been demonstrated for the transcription factor, Pit-1,associate with critical cofactors such as MDM2, TAFs, which can switch from an activator to a repressor by aand TRAPs (Cain et al., 2000 and references therein). two base pair change in its binding sites (Scully et al.,

Seminal experiments by Kim and colleagues demon- 2000). For this reason, the use of an optimized consen-strated that cruciforms or other non-B-DNA structures sus DNA sequence for p53 interaction may not be appro-are normal recognition elements for p53 (Kim et al., 1997, priate because it does not represent the physiological1999). This would explain why unmodified p53 fails to complexity of p53 function.bind to a short DNA oligonucleotide in EMSA, because It will be of great interest in the future to examine thethis sequence cannot readily form the secondary struc- distinct mechanisms employed by p53 to choose amongture it would usually adopt in a larger piece of DNA. its numerous target genes to regulate specific pathwaysThus, modification by acetylation, antibody association, in response to cellular stress.or deletion of the C terminus might be required for p53

Experimental Proceduresto bind to a DNA structure that the native protein nor-mally does not recognize. This interpretation by Kim et

Plasmids and Expression of Recombinant Proteinsal (1997, 1999) is entirely consistent with our results.The WWP-LUC (p21-LUC) plasmid was constructed as described

More recently, it was demonstrated that p53 can be in El-Deiry et al. (1995) and was a generous gift of Dr. Bert Vogelstein.specifically directed to cruciform structures, even though Full-length human Flag-tagged p53 was expressed in Sf9 cells andthe sequences forming the cruciform do not fit the p53 purified from total cell extracts by affinity to anti-Flag M2 affinity

gel (Sigma) according to the protocol used to purify Flag-taggedconsensus binding site (Jett et al., 2000). Taken to-dACF described by Ito et al. (1999). p53 baculovirus was a gift fromgether, these studies emphasize the importance of usingDr. Ingrid Grummt and Dr. Renate Voit. Baculovirus for expressionDNA recognition sequences within an appropriate con-of the C-terminal deletion mutant p53 �C30 (amino acid 1–353) wastext when examining the effect of both C- anda generous gift from Dr. Carol Prives, and the protein was purified

N-terminal modifications on p53 binding. Moreover, the from Sf9 cells as described by Cain et al. (2000). cDNA encoding theresults may differ depending upon the specific target p53KR was obtained from Dr. Wei Gu, subcloned into baculovirusgene and p53 recognition sequence used. expression vector (Bac-to-Bac System, Gibco BRL), and the protein

was purified by similar means. Recombinant ACF complex (Flag-It is intriguing that p53 binds to the p21 promoter withtagged dACF � untagged dISWI) was produced in Sf9 cells andhigher affinity and with different kinetics when assem-purified by anti-Flag M2 (Sigma) affinity chromatography as de-bled into chromatin than it does to DNA. Particularlyscribed by Ito et al. (1999). Histidine-tagged NAP-1 was purified

because this occurs in the absence of chromatin remod- from baculovirus-infected Sf9 cells by Ni-NTA (Qiagen) affinity chro-eling or modifying complexes and is not observed with matography, followed by a conventional Mono-Q chromatographicother transcription factors that can also bind to nucleo- step. Full-length human Histidine-tagged p300 was expressed in

Sf9 cells and purified as previously described by Kraus et al. (1999).somes, such as Gal4-VP16 (Pazin et al., 1998). This couldACF, NAP-1, and p300 baculoviruses were a generous gift of Dr.be explained if bending of the DNA, when wrappedJames Kadonaga.around a nucleosome, generates a secondary structure

that is more stable for p53 binding. Indeed, previousElectrophoretic Mobility Shift Assay

studies determined the importance of DNA bending for Acetylation of p53 by p300 was performed as described by Gu andp53 high-affinity binding and predicted that some p53 Roeder (1997). EMSA was carried out essentially as described bybinding sites would be exposed and accessible when Jayaraman, et al. (1998).The 25 bp double-stranded oligonucleotide

harboring the 5� site of the p21 promoter was generated by annealingincorporated into a nucleosome (Nagaich et al., 1999).the single-stranded oligonucleotides 5�-caggaacatgtcccaacatgttImportantly, our findings demonstrate that the structuregaa-3� and 5�-ttcaacatgttgggacatgttcctg-3�. The 160 bp region ofrecognized by p53 in p21 promoter DNA is preservedthe p21 promoter containing a centered 5� site was generated byand improved or stabilized in chromatin. The physiologi-PCR from the plasmid p21-LUC, using the primers 5�-cgggctgcag

cal significance of the distinct kinetics of p53 occupancy gaattcgatatc-3� (sense) and 5�-ccatccccttcctcacctgata-3� (anti-observed on the p21 promoter as chromatin or DNA sense). Both probes were 32P end-labeled using T4 polynucleotideis unclear. The linear rate of association of p53 with kinase and further purified by 12%–15% polyacrylamide native gels.chromatin may indicate that lower concentrations of p53

Chromatin Assembly and In Vitro Transcriptionare required to fully occupy binding sites in vivo thanChromatin was reconstituted using Drosophila embryonic extractsthe cooperative binding to DNA would indicate. Thisas described (Bulger and Kadonaga, 1994). After assembly, thecould be significant if the cell has to respond efficientlychromatin template (500 ng of DNA in 50 l) was incubated withto activate p53-responsive pathways without waiting forp53 and/or p300 (typically between 0.2–10 pmol of p53 and 0.2–0.5

a critical threshold of p53 concentration to be reached. pmol of p300), for 30 min at 27C. For transcription, 20 l of HeLaIt should be emphasized, however, that the nature of cell nuclear extract (typically 8 mg/ml) was added and incubatedp53 binding to chromatin and the requirements for re- on ice for 10 min, then reactions proceeded as described (Kadam

et al., 2000). The purified RNA was analyzed by primer extensionmodeling/modifying activities may vary with individualanalysis with primers specific to luciferase and �-globin gene (usedtarget promoters.as an internal control) sequences.One can surmise that the variable nature of DNA con-

sensus sequences recognized by p53 is a critical regula-DNase I Footprinting of Chromatin and DNA

tory feature, because it enables diverse structures to be DNase I footprinting was done using three different templates: crudegenerated within target promoters and manipulated by chromatin-assembled mixtures, CL4B-purified chromatin, and na-a variety of cellular signals. In fact, the ability of p53 to ked DNA. For purification of chromatin assembled with Drosophila

embryonic extracts, we proceeded as previously described (Mizu-function as an activator or repressor and differentially

Molecular Cell68

guchi and Wu, 1999). After incubation of the different templates with El-Deiry, W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R.,Trent, J.M., Lin, D., Mercer, W.E., Kinzler, K.W., and Vogelstein, B.the proteins as indicated in Figure legends, samples were digested

with 33 U/ml (crude chromatin), 1.5 U/ml (purified chromatin) or 0.1 (1993). WAF1, a potential mediator of p53 tumor suppression. Cell75, 817–825.U/ml (naked DNA) DNase I (Boehringer Mannheim) for 75 s at 25C.

Purified DNA fragments were analyzed by primer extension. El-Deiry, W.S., Tokino, T., Waldman, T., Oliner, J.D., Velculescu, V.E.,Burrell, M., Hill, D.E., Healy, E., Rees, J.L., Hamilton, S.R., et al.

Western Blot Analysis (1995). Topological control of p21WAF1/CIP1 expression in normalFor Western blot analysis of chromatin samples, mixtures were and neoplastic tissues. Cancer Res. 55, 2910–2919.boiled for 15 min in SDS-PAGE loading buffer and resolved by 10%

Gu, W., and Roeder, R.G. (1997). Activation of p53 sequence-specificSDS-PAGE electrophoresis. After transfer of proteins to PVDF mem-

DNA-binding by acetylation of the p53 C-terminal domain. Cell 90,branes, filters were blocked and incubated with monoclonal anti-

595–606.body DO-1 (unmodified N-terminal domain of p53, Oncogene Re-

Hupp, T.R., Meek, D.W., Midgley, C.A., and Lane, D.P. (1992). Regu-search Products), polyclonal antibodies PC198 (acetylated p53,lation of the specific DNA-binding function of p53. Cell 71, 875–886.Oncogene Research products), and polyclonal antibodies C20

(p300, Santa Cruz Biotechnology). Blots were developed using Ito, T., Ikehara, T., Nakagawa, T., Kraus, W.L., and Muramatsu, M.Amersham-enhanced chemiluminesence reagents. (2000). p300-mediated acetylation facilitates the transfer of histone

H2A–H2B dimers from nucleosomes to a histone chaperone.Genes & Dev. 14, 1899–1907.Chromatin HAT Assay

Chromatin assembly was performed with recombinant dACF com- Ito, T., Levenstein, M.E., Fyodorov, D.V., Kutach, A.K., Kobayashi,plex and NAP-1 histone chaperone as described by Ito et al. (1999) R., and Kadonaga, J.T. (1999). ACF consists of two subunits, Acf1and references therein. Chromatin assembly was assessed by mi- and ISWI, that function cooperatively in the ATP-dependent cataly-crococcal nuclease digestion and transcriptional repression. After sis of chromatin assembly. Genes & Dev. 13, 1529–1539.assembly, proteins were added to the chromatin mixtures in the

Jayaraman, L., Moorthy, N.C., Murthy, K.G., Manley, J.L., Bustin,presence of 5 M 3H-acetyl CoA (ICN). After incubation for 1 hr at

M., and Prives, C. (1998). High mobility group protein-1 (HMG-1) is30C with p53 and/or p300, reactions were stopped by boiling in

a unique activator of p53. Genes & Dev. 12, 462–472.SDS-PAGE loading buffer and analyzed on a 15% SDS-PAGE gel.

Jett, S.D., Cherny, D.I., Subramanian, V., and Jovin, T.M. (2000).The gel was fixed and treated with fluorography enhancing solutionsScanning force microscopy of the complexes of p53 core domain(NEN Life Science). The gel was then dried and exposed to autoradi-with supercoiled DNA. J. Mol. Biol. 299, 585–592.ography.Kadam, S., McAlpine, G.S., Phelan, M.L., Kingston, R.E., Jones, K.A.,

Chromatin Immunoprecipitation Assay and Emerson, B.M. (2000). Functional selectivity of recombinantChromatin immunoprecipitation analysis was performed using the mammalian SWI/SNF subunits. Genes & Dev. 14, 2441–2451.anti-acetylated histone H4 antibody (Upstate Biotechnology) ac- Kim, E., Albrechtsen, N., and Deppert, W. (1997). DNA-conformationcording to the manufacturer’s recommendations and as previously is an important determinant of sequence-specific DNA-binding bydescribed (Kundu et al. 2000) with some minor modifications. tumor suppressor p53. Oncogene 15, 857–869.

Kim, E., Rohaly, G., Heinrichs, S., Gimnopoulos, D., Meissner, H.,Acknowledgments

and Deppert, W. (1999). Influence of promoter DNA topology onsequence-specific DNA-binding and transactivation by tumor sup-

We thank Drs. Bert Vogelstein, Renate Voit, Ingrid Grummt, Carolpressor p53. Oncogene 18, 7310–7318.

Prives, Wei Gu, and James Kadonaga for their generous gifts ofKraus, W.L., Manning, E.T., and Kadonaga, J.T. (1999). Biochemicalreagents used in these studies. We also thank Erich Ehlert andanalysis of distinct activation functions in p300 that enhance tran-Gordon Pfeifer for their expert technical assistance. This work wasscription initiation with chromatin templates. Mol. Cell. Biol. 19,supported by grants to B.M.E. from the National Institutes of Health.8123–8135.J.M.E. is supported by the Pew Latin American Fellows Program in

the Biomedical Sciences sponsored by the Pew Charitable Trusts. Kundu, T.K., Palhan, V.B., Wang, Z., An, W., Cole, P.A., and Roeder,R.G. (2000). Activator-dependent transcription from chromatin invitro involving targeted histone acetylation by p300. Mol. Cell 6,Received March 22, 2001; revised May 25, 2001.551–561.

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