p53 binding to nucleosomes within the p21promoter in vivo leads to nucleosome lossand transcriptional activationOleg Laptenko, Rachel Beckerman, Ella Freulich, and Carol Prives1

Department of Biological Sciences, Columbia University, New York, NY 10027

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2008.

Contributed by Carol Prives, April 26, 2011 (sent for review November 17, 2010)

It is well established that p53 contacts DNA in a sequence-dependent manner in order to transactivate its myriad targetgenes. Yet little is known about how p53 interacts with its bindingsite/response element (RE) within such genes in vivo in the contextof nucleosomal DNA. In this study we demonstrate that both distal(5′) and proximal (3′) p53 REs within the promoter of the p21 genein unstressed HCT116 colon carcinoma cells are localized within aregion of relatively high nucleosome occupancy. In the absence ofcellular stress, p53 is prebound to both p21 REs within nucleosomalDNA in these cells. Treatment of cells with the DNA-damaging drugdoxorubicin or the p53 stabilizing agent Nutlin-3, however, isaccompanied by p53-dependent subsequent loss of nucleosomesassociated with such p53 REs. We show that in vitro p53 can bindto mononucleosomal DNA containing the distal p21 RE, providedthe binding site is not close to the diad center of the nucleosome.In line with this, our data indicate that the p53 distal RE within thep21 gene is located close to the end of the nucleosome. Thus, low-and high-resolution mapping of nucleosome boundaries aroundp53 REs within the p21 promoter have provided insight into themechanism of p53 binding to its sites in cells and the consequentchanges in nucleosome occupancy at such sites.

DNA binding ∣ DNA damage

In eukaryotic cells, genomic DNA is tightly associated with his-tones resulting in the organized and dynamic structure known as

chromatin (1, 2). The primary unit of chromatin is the nucleo-some, which is composed of approximately 146 bp DNA woundaround the core histone octamer (3). The resulting higher-ordered structure helps to compact the DNA within the nucleus.At the same time it represents an accessibility barrier for specifictranscription factors whose primary role is to regulate the multi-step process of transcriptional activation of most gene promotersin response to pathway-activating stimuli. Many in vitro biochem-ical studies have revealed significant reduction in transcriptionfactor binding affinities toward their cognate sites within nucleo-somal DNA as compared to naked DNA. In vivo, a number ofmolecular mechanisms may promote specific and efficient inter-actions between a given transcriptional regulator and its bindingsite within DNA. Some of these mechanisms depend on theenzymatic activities of chromatin remodeling complexes that fa-cilitate either nucleosome eviction or sliding (4), while others relyon cooperative binding between transcription factors (5), localhistone modifications (6), and/or prior nucleosome interactionswith so-called “pioneer” factors (7).

p53 is a sequence-specific transcriptional activator that exertsits tumor-suppressor activity primarily through regulation oftranscription initiation of multiple downstream target genes (8).Mutations within the DNA-binding domain (DBD) of p53 andsubsequent loss of specific DNA-binding activity are responsiblefor p53 inactivation in more than 50% of tumors. The p53 con-sensus binding site is quite complex and consists of two decamerichalf-sites, RRRCA/TT/AGYYY that are usually directly adjacent

but can be separated by up to 13 bp (9–11). Multiple studies inrecent years have focused on the interaction of p53 with itscognate binding sites in vivo and in vitro and subsequent genetransactivation (or transrepression). Here we have examined thenucleosomal status in vivo of p53 binding sites within one of itsmajor target genes, p21, before and after induction of p53 andhave also determined the extent to which p53 is able to interactwith its cognate sites within nucleosomal context.

Resultsp53-Dependent Loss of Nucleosomes Occurs at p53 Binding SitesWithin the p21 Promoter.We focused on p21 as it is one of the bestcharacterized bona fide p53 target genes. The p21 promoterhas two p53 binding sites (or response elements, REs) that con-form to the p53 consensus binding sequence (Fig. 1A), the moredistal (5′) site at −2283 that binds p53 relatively strongly and themore proximal (3′) site at −1391 that is more weakly bound byp53 (12–14). We first determined the nucleosome status at thesesites between matched HCT116 colon carcinoma cell lines thateither contain (þ∕þ) or lack (−∕−) full-length p53 (15). As ex-pected, treatment with doxorubicin (dox) resulted in a significantincrease of p53 levels in HCT116 (þ∕þ) cells and this precededincreases in both p21mRNA and protein (Fig. S1 in SI Appendix).Activation of p21 expression correlated with p53 binding to its 5′and 3′ REs as measured by chromatin immunoprecipitation(ChIP) (Fig. 1B). Consistent with the known higher affinity ofp53 for the 5′ RE than for the 3′ RE, there was significant basalbinding to the former site that was even equivalent to binding tothe 3′ site after 4 h of dox treatment. To examine the nucleosomestatus of the p53 REs within the p21 promoter, we determined theextent to which these regions were resistant to micrococcal nu-clease (MNase) digestion as reported for other transcribed genes(16). Cells were treated or not with dox followed by cross-linkingwith formaldehyde (to preserve chromatin structure) and thennuclei were isolated and incubated with MNase. DNA recoveredfrom the MNase-resistant mononucleosomal fraction wasextracted from the gel and subjected to Q-RT-PCR analysis toassess the nucleosome status in the vicinity of the two p53REs in the p21 promoter region as well as in two control regions:the TATA box at −20 bp and further downstream þ11.4 kb. Inunstressed HCT116 (þ∕þ) or (−∕−) cells both p53 REs had arelatively high nucleosome content when compared to the TATAregion that was previously reported to be bound by RNA poly-merase II and virtually nucleosome free (17) (Fig. 1C, time “0”).Notably, dox treatment resulted in a rapid p53-dependent loss ofnucleosomal content within both p53 REs (Fig. 1 C and D), while

Author contributions: O.L. and C.P. designed research; O.L., R.B., and E.F. performedresearch; O.L., R.B., and C.P. analyzed data; and O.L. and C.P. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence may be addressed. E-mail: clp3@columbia.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1105680108/-/DCSupplemental.

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we detected no p53-associated increase in MNase sensitivity with-in any of the two control regions (Fig. 1E).

Because dox can induce DNA strand breaks and thereby acti-vate a number of intracellular processes associated with chroma-tin remodeling independently of p53 (18), we performed thesame experiment using Nutlin-3 that disrupts the p53 interactionwith Mdm2 and thereby stabilizes p53 in the absence of DNAdamage (19). Nutlin-3 induced p21 RNA accumulation thatwas accompanied by a decrease in MNase-resistant DNA at bothp53 REs (Fig. S2 A–C in SI Appendix). The kinetics of nucleo-some loss from both p53 REs upon treatment with either p53stabilizing agent was very similar. Note that dox-induced loss ofnucleosomes within the vicinity of the 5′ RE in HCT116 (þ∕þ)cells required the presence of p53, because introduction of p53siRNA but not control siRNA into these cells prior to drug treat-ment resulted in significant loss of MNase hypersensitivity withinthat region (Fig. S3 in SI Appendix).

We asked whether the altered nucleosome occupancy at thep53 REs depended upon ongoing transcription of p21 RNA byperforming a similar experiment to the one shown in Fig. 1 butin the presence or absence of the potent RNA Polymerase II in-hibitor, α-amanitin (Fig. S4 in SI Appendix). While transcriptionof several p53-dependent targets such as p21, puma, and mdm2was blocked by α-amanitin regardless of p53 activation status(Fig. S4A in SI Appendix), MNase hypersensitivity within thep21 REs remained unchanged by the inhibitor (Fig. S4B in SIAppendix). Thus, p53 does not require active transcription tooccur in order for it to initiate eviction of nucleosomes within theregions of its binding sites.

Localized p53-Dependent Eviction of Nucleosomes Occurs at the p53REs Within the p21 Promoter. We extended our analysis of the nu-cleosome density changes within chromatin at the p53 REs ofthe p21 promoter after dox treatment. For this purpose, multipleprimer pairs were designed covering 450–500 bp surroundingeither the distal or proximal p53 REs (Fig. 2A and Fig. S5A inSI Appendix, respectively). The length of an average ampliconobtained in a Q-PCR reaction with either primer pair wasapproximately 85 bp and sequences of neighboring ampliconsoverlapped by approximately 10 bp. Unfortunately, we couldnot design primers that would cover approximately 60 bp gapsthat were 5′ to either the distal or the proximal site (between thefirst and second primer pairs; see Fig. 2A and Fig. S5A in

SI Appendix). For analysis of those, we employed the ligation-mediated (LM) PCR technique (see Fig. 5 below).

Unexpectedly, this low-resolution mapping experiment re-vealed the presence of two distinct (though closely spaced) sitesof MNase sensitivity within the distal p53 RE locus in HCT116þ∕þ) cells (Fig. 2 B–D). The first coincides with the original p53RE itself (primer pairs 2 and 3) and the second site is locatedabout 150–160 bp 3′ to the RE (primer pair 5). These alterationsrequired full-length p53 because there were no significantchanges within the corresponding regions in HCT116 −∕− cells(Fig. 2C). In HCT116 (þ∕þ) cells the relative increases in MNasesensitivity upon treatment with dox were accompanied by asignificant loss of histones within both regions as shown byan MNase-assisted ChIP experiment (Fig. 2D). Interestingly,between these two hypersensitive regions we also detected a sitethat became more resistant to MNase under conditions thatactivate p53, and MNase-assisted ChIP analysis of that regionrevealed that it, too, had a relative increase in local histoneoccupancy (primer pair 4).

Although p53 binds far more weakly to the proximal RE in thep21 promoter, the MNase sensitivity profile within that regionwas very similar to that seen at the stronger distal RE element(compare the data from Fig. 2B with Fig. S5B of SI Appendix).This region showed two MNase-sensitive sites (primer pairs 2,3, and 6) separated by a region where there was no significantchange in MNase-resistant DNA (primer pairs 4∕5). There werealso corresponding changes in histone H3 occupancy within thisregion. As with the 5′RE region, there were only very modestdox-dependent changes in nucleosome distribution at thep21 proximal RE in the absence of full-length p53 (Fig. S5C inSI Appendix).

p53 Is Bound to Nucleosomal DNA in Unstressed HCT116 Cells.Although the above mapping experiments elucidated featuresof the chromatin organization within the distal and proximalp53 REs, they did not reveal whether p53 can bind directly tomononucleosomal DNA in vivo. To address this mononucleo-somes prepared from formaldehyde-crosslinked chromatin bylimited digestion with MNase were immunoprecipitated witheither anti-p53 or anti-H3 antibodies. The recovered mononu-cleosomal DNA was amplified by Q-PCR using primer pairs spe-cific to the p21 p53 distal and proximal REs, or the two negativecontrol regions at the TATA and þ11443 regions (Fig. 3). Theresults strongly indicate that in unstressed cells p53 is bound

Fig. 1. Doxorubicin treatment leads to p53-depen-dent loss of nucleosomes at p53 binding sites withinthe p21 promoter. (A) p21 promoter region showingp53 5′ and 3′ REs, TATA region and þ11.4 kb controlregion. Bold arrow represents the p21 transcriptionstart site and head-to-head oriented pairs of arrowsrepresent the sites that were assayed for MNase hy-persensitivity by Q-RT-PCR amplification. (B) HCT116(þ∕þ) cells were incubated with 0.75 μM dox fol-lowed by processing for ChIP analysis of p53 bindingto p21 5′ and 3′ REs and þ11.4 kb region as a nega-tive control. (C) HCT116 (þ∕þ) cells were treated with0.75 μM dox for indicated time periods as above, fol-lowed by formaldehyde cross-linking and processingto determine relative mononucleosomal occupancyat the p21 distal 5′ and proximal 3′ p53 REs as wellas the p21 TATA region. MNase-digested chromatinwere deproteinized and mononucleosomal DNAwas gel-purified for use as templates in Q-RT-PCRwith pairs of primers flanking indicated regions with-in the p21 promoter. (D) Same as in C performedusing HCT116 p53 (−∕−) cells. (E) MNase hypersensi-tivity data obtained for two control regions withinp21 gene, with relatively high (11.4 kb site), andlow (TATA) initial nucleosomal content.

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to MNase-resistant DNA and, depending on the site, such bind-ing is either greatly decreased or completely lost upon treatmentwith dox, which is mirrored by significant loss of histones withinthe p53 REs. Supporting this likelihood, the signal at the REs was

markedly greater than those with either the TATA or þ11443 re-gions where there was also very little change after dox treatment.Note that while the amount of specific mononucleosomal DNAimmunoprecipitated with the p53 antibodies was reduced upondox-dependent p53 activation (as measured by MNase-assistedChiP), total p53 binding to the p21 REs was increased over thesame time period after dox treatment (compare the data fromFig. 3 and from Fig. 1B). Thus, loss of the signal upon p53 activa-tion in the MNase-assisted ChiP assay simply reflects the loss ofnucleosomes but not the loss of overall p53 DNA-binding per se.

Stable Nucleosomal Binding by p53 Requires That Its Site Be Close tothe End of Nucleosomal DNA. With few exceptions biochemicalstudies on p53/DNA interactions have been examined in the con-text of naked DNA. We therefore sought to recapitulate our ob-servations showing that p53 binds to its sites within chromatin atthe p21 promoter by examining how p53 interacts with mononu-cleosomes in vitro. We performed DNase I footprinting usingeither naked or reconstituted mononucleosomal DNA spanningthe p21 promoter p53 REs (Fig. S6 in SI Appendix). A strong nu-cleosome positioning sequence derived from the yeast HSP82promoter region was incorporated into a chimeric 170 bpDNA construct that had the p21 distal p53 RE located 30 bp fromthe 5′-end (see SI Materials and Methods in SI Appendix for de-tails). As expected, preincubation of naked DNA with increasingamounts of purified wild-type p53 protein resulted in strong pro-tection from DNase I cleavage within the RE region (Fig. S6A inSI Appendix). As described previously (20), when the same DNAwas present within a mononucleosome, DNase I cleavage yieldeda characteristic approximately 10-bp periodicity pattern thatrevealed the contacts between the DNA and the core histone oc-tamer (Fig. S6B in SI Appendix). p53 binding to this mononucleo-somal DNA resulted in clear but relatively weak protection of thep21 gene distal RE (see densitometry analysis) along with anappearance of a DNase I hyper-sensitive site located at the 3′edge of the RE sequence (indicated by asterisk).

An electrophoretic mobility shift assay (EMSA) was also per-formed to characterize p53 interactions with mononucleosomalDNA in vitro (Fig. 4). Our initial experiments revealed thatp53 did not detectably bind to such DNA from the p21 promoterwhen the p53 5′ RE was centrally positioned (Fig. S7 in SIAppendix) so we assessed whether p53 might bind to its site atother positions within mononucleosomal DNA. To this end, weprepared a set of ten 170-bp chimeric DNA templates containingthe p21 p53 5′ RE located at positions that differed by one base

Fig. 2. Doxorubincin treatment alters nucleosome distribution near the p21distal p53 binding site. (A) Schematic representation of the approximately500-bp region surrounding the p21 5′ p53 RE together with the correspond-ing amplicons. Each filled segment represents 20 bp and the open segmentis the location of the 5′ p53 RE. Note that there is an approximately 60-bpgap between the first and the second amplicons that could not be assayed todue to difficulties with primer design. (B and C) MNase-resistant DNA in theamplified regions shown in A. Mononucleosomal DNA was prepared asdescribed in the SI Materials and Methods in SI Appendix from eitherHCT116 (þ∕þ) (B) or HCT116 (−∕−) (C) cells treated with 0.75 μM doxorubicinfor 0 (filled bars) or 8 h (open bars). (D) MNase-assisted ChIP (see SI Materialsand Methods in SI Appendix) was used to measure the relative amounts ofH3 histone associated with each amplicon in A in HCT116 (þ∕þ) cells. Dataare presented as the ratio of histone H3 at 8 hr post Dox/ histone H3 at 0 hr(untreated).

Fig. 3. p53 is bound to nucleosomal DNA in cells prior totreatment with doxorubicin. MNase-assisted ChIP was per-formed using HCT116 (þ∕þ) cells treated with 0.75 μM dox-orubicin for 0 (filled bars) or 8 (open bars) hours. Q-RT-PCRwas used to measure MNase-resistant DNA immunoprecipi-tated with either anti-p53 or anti-H3 antibodies as indi-cated at (A) p21 5′ p53 RE, (B) p21 3′ p53 RE, (C) p21 TATAregion, or (D) p21 þ11.4 region. Dotted line represents thebackground signal for each experiment.

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pair in their distance from the 5′-end of the DNA (Fig. 4A).EMSA analysis showed that p53 binding to mononucleosomesbecame detectable and increased as the RE was moved furtherfrom the center of the DNA. In fact, a 2-bp shift in the RE(170–30) resulted in an approximately fivefold increase in bindingto mononucleosomal DNA when compared to a mononucleo-some where the RE was located 32 bp from the end of the DNA(170–32), and a 9-bp shift in the RE toward the center (170–39)reduced binding by more than a factor of 10 (see Fig. 4 B and C,graph). Thus, the distance between the binding site and the end ofthe DNA represents a critical parameter influencing binding ofp53 to mononucleosomal DNA. A similar dependence of bindingto the site within nucleosomal DNA was demonstrated before forother sequence-specific proteins (e.g., for restriction endonu-cleases) (21).

Mapping the Location(s) of the Distal p53 Binding Site Within a Nucleo-some in the p21 Promoter in Vivo.We next sought to determine thelocation of the p21 distal p53 binding site within the relevantmononuclesome in vivo by performing linker-mediated PCR(LMPCR) in HCT116þ ∕þ cells (Fig. 5). This analysis revealedseveral major primer extension products (numbered 1–5) locatedat different distances from the p53 RE that we interpret as repre-senting nucleosomal boundaries. In Fig. 5 products numbered 1and 2, that are 107 and 68 bp from the p53 5′ RE, respectively,likely represent the boundaries of two neighboring nucleosomes.Additionally, three products of weaker intensity (numbered 3, 4,and 5) were situated much closer to the p53 RE at 41, 32, and18 bp, respectively. Indeed, consistent with our in vitro bindingdata, the location of these products positions the p53 binding siteclose to the edge of the nucleosome, thus making it more acces-sible for p53. Further, all above-mentioned species were reducedupon dox treatment, which is in good agreement with the dataobtained from the low-resolution mapping experiment shownin Fig. 2B. This pattern of LMPCR extension products suggeststhat the nucleosome at the distal p53 RE is somewhat heteroge-neously positioned. We assume that formaldehyde cross-linkingprior to MNase treatment helped us to catch a nucleosome thatcontains the p53 distal RE in unstressed cells in several most-probable positions (i.e., positions 2, 3, 4, and 5). Analysis ofthe proximal 3’ p53 RE by LMPCR also revealed multiple species

(the most prominent one situated about 55 bp away from the RE)that again likely represent multiple potential nucleosome bound-aries (Fig. S8 in SI Appendix). Here too there was a significantloss of intensity of the major band/boundary and almost totalvanishing of minor bands 8 h after administration of dox.

Discussionp21 was one of the first genes found to be positively regulated byp53 (12, 22). Although a number of studies have addressed thebinding of p53 to its distal and proximal binding sites within thep21 promoter in vitro and in vivo, none has interrogated suchbinding in the context of mononucleosomal DNA in detail. In thisstudy we have gained insight into chromatin organization in thep21 promoter, in particular within regions spanning its two p53REs. By applying ChIP, MNase hypersensitivity assays, MNase-assisted ChIP, and LM PCR, we demonstrate that p53 REs withinthe p21 promoter in unstressed HCT116 cells are localized withinnucleosomal DNA. Further, nucleosomes are rapidly lost uponactivation of p53 with either the DNA-damaging agent doxoru-bicin or by blocking its degradation by Mdm2 through adminis-tration of Nutlin-3. Changes in hypersensitivity within both p53REs of p21 promoter may be explained by either nucleosomesliding or nucleosome eviction. After p53 activation by dox, with-in the vicinity of the p21 5’ RE there is an apparent loss of twonucleosomes (Fig. 2, amplicons 2 and 5) and between them thereis a relative increase in H3 occupancy within amplicon 4. Yet thedistance covered by amplicon 4 is not long enough to contain afull nucleosome core particle (i.e., consisting of the cannonicalhistone octamer). Conceivably partial loss of a portion of the his-tones from that nucleosome and/or a subsequent clash betweentwo neighboring nucleosomes has occurred. This is not unheardof—see, for example, Dechassa et al. (23). Using in vitro recon-stituted mono-, di-, and three-nucleosome arrays in the presenceof SWI/SNF, those authors demonstrated collision of two neigh-boring nucleosomes, as well as partial loss of H2A/H2B dimer. Asdiscussed below, the presence of certain histone variants (H3.3and H2A.Z, in particular) and their effect on nucleosome stabilitymay also be in play here.

Does p53 bind to nucleosomal DNA and cause loss of nucleo-somes (directly by destabilizing multiple histone-DNA contacts,or indirectly by bringing down components of chromatin remo-deling machinery), or does nucleosome loss precede and is neces-

Fig. 4. Detectable p53 binding to the p21 distal RErequires the site to be positioned close to the end ofa mononucleosome. (A) The location of p21 distalp53 REs inserted within 170 BP DNA fragments fromyeast HSP82 sequence containing a strong position-ing sequence assembled into mononucleosomal DNAby the octamer transfer method. (B) An electro-phoretic mobility shift assay was performed with[32P] labeled mononuclesomal DNAs shown in Aand purified p53 protein. Four different DNA/mono-nucleosomes were run on one gel, and then con-struct (170–30) was re-run on each gel to correctdifferences between the experiments. Shown arePhosphorImager scans of three 4% native 0.5X TBEgels with 10 mononucleosomal constructs. Phosphor-Imager scans in B were analyzed using ImageQuantSoftware and presented as graphs (C).

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sary for p53 sequence-specific binding? The necessity of nucleo-some removal for gene activation was suggested more than twodecades ago. Since then, numerous in vitro binding experimentsperformed on reconstituted nucleosomal substrates have almostexclusively supported the idea that efficient binding of a giventranscription factor to its RE is greatly reduced in the contextof a nucleosome. Factors that were shown to interact with nucleo-somes (e.g., TFIIIA, Sp1) have significantly reduced bindingaffinities when compared to naked DNA (5). Given the complex-ity of p53 REs and multiplicity of contacts between the p53core domain and DNA sequences within each RE (24, 25), weassumed that p53 would not bind detectably to its sites whenwrapped around the histone octamer. Surprisingly, our MNase-assisted ChIP experiment performed on p21 distal and proximalREs demonstrated that p53 in unstressed cells is bound to nucleo-somal DNA. What is the significance of such binding whenp53 becomes activated by stress signals? We envision two possiblescenarios. In the first, increasing amounts of stabilized p53 tran-siently compete with a nucleosome for the RE. In this case, whenp53 levels are increased (e.g., after DNA damage) the relativelylow affinity of p53 for its binding site within the nucleosomemightstill be enough to result in efficient transcriptional activation invivo. Indeed, recent experiments using fluorescence recoveryafter photobleaching (FRAP) have revealed that the nature oftranscription factor (TF) binding to chromatin is very transient,and the time that a given TF spends on its RE in vivo may beless than a minute (reviewed in ref. 26). This time frame may besufficient to bring down the components of modifying and/orremodeling machinery leading to local nucleosome eviction/dis-placement, thereby culminating in formation of a competent pre-initiation complex and subsequent activation of transcription.Experiments that employ FRAP and/or single molecule technol-ogy may provide estimates of the half-life of p53 bound to its REswithin nucleosomes or chromatin.

The second scenario depends on the ability of nucleosomes toadopt multiple positions within certain regions, which is dictatedboth by the DNA sequence and components of the chromatinremodeling machinery (reviewed in ref. 27). In this case, initialp53 binding will occur under the most favorable conditions, such

as positioning of the RE in relatively close proximity to the end ofthe nucleosome. Indeed, our LM PCR and EMSA experimentssupport this theory. Interestingly, large scale genome analysis ofdata on the distribution of nucleosomes within the p21 promoterin either A375 or MDA-kb2 cell lines indicates that both p53REs are situated in regions with fading nucleosomal density, veryclose to a relatively nucleosome-depleted part of the promoter(UCSC Genome browser on Human, March 2006 Assembly;NCBI36/hg18). Our low- and high-resolution mapping experi-ments correlate well with these findings. We acknowledge thatnucleosomal profiles differ between cell lines, which could berelated to changes in DNA such as mutations, deletions, or am-plifications, as well as epigenetic changes, all of which may con-tribute to the chromatin landscape within a given cell, tissue ororganism.

In addition to showing that p53 is able to bind to nucleosomalDNA both in vitro and in vivo, our study raises the possibility of apotential second p53 binding site that would reside approximately150–160 bp downstream of the bona fide distal p21 RE (Fig. 2,primer pair 5). This is supported by increased p53-dependentMNase sensitivity and p53-dependent loss of core histones aswell as the presence of three overlapping weak p53 half-siteswithin that region. Whether the putative second binding regionexists and is functional in vivo remains to be answered by futureexperiments.

Local histone modifications and/or histone variants may alsoaffect p53 binding to nucleosomes. For example, the histone H2variant, H2A.Z, is enriched at p53 REs within the p21 promoterand DNA-damage stress caused by doxorubicin leads to evictionof H2A.Z in a p53-dependent but transcription-independentmanner (28). The presence of H2A.Z may positively influencep53 binding to mononucleosomes because the H2A.Z/H2Bhistone dimer is less stable than the regular H2A/H2B dimerand can be relatively easily released from nucleosomes (29).Experiments involving in vitro p53 binding to the RE localizedwithin mononucleosome reconstituted with either H2A orH2A.Z may provide insight into the relative affinities of such sub-strates toward p53.

Fig. 5. High-resolution mapping of MNase-sensitive sitesindicate that the p21 distal p53 binding site is close tothe end of a nucleosome in vivo. HCT116 (þ∕þ) p53 cellswere treated with 0.75 μM doxorubicin for 0 or 8 hoursafter which cells were cross-linked with formaldehyde fol-lowed by isolation of nuclei and treatment with three dif-ferent concentrations of MNase. Deproteinized, purifiedDNA was subjected to LM PCR as described in SI Materialsand Methods in SI Appendix. Left panel shows PhosphorI-mager scan of the 9% polyacrylamide gel that resolvedthe LMPCR products.AGCT lanes are DNAmarkers obtainedin primer extension reactions with acyclo-ATP, -GTP, -CTP,and -TTP, respectively (New England Biolabs). Graph onright shows densitometry analysis of the gel products at0 and 8 hours after dox treatment. Putative nucleosomeboundaries deduced from MNase-sensitive sites and theirlocations relative to the p53 5′ RE are depicted below.

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A different mechanism for p53-nucleosome interaction wasproposed by Sahu et al. based on the results of an elegant in vitrostudy (30). The authors suggest that orientation of the p53 bind-ing site on a nucleosome in conjuction with a nucleosomal posi-tioning sequence may preset the p53 RE in a way that is easilyaccessible to p53. Though attractive, confirmation of this modelrequires support from experiments on p53 binding to bona fideREs in the context of their natural settings in vivo.

Certain p53 REs may localize within nucleosome-depleted re-gions. In these cases p53 binding may be a different and less com-plex process and could be regulated by different mechanism(s)such as relative strength of the RE, local concentrations of p53,modification status of p53, or cooperative binding with othertranscription factors. Indeed, the results of MNase hypersensitiv-ity analysis performed on several p53-dependent promoters inHCT 116 þ∕þ and −∕− cells rather support this assumption(Fig. S9 in SI Appendix). The levels of basal nucleosome occu-pancy differ drastically at p53 REs within the mdm2P2, bax,puma, or PCNA promoters: Whereas the first two are virtuallynucleosome free, bax and (even more so) puma p53 REs showstrong MNase resistance. Interestingly as well, there is relativelyhigher basal nucleosome occupancy detected within p53 REs ofthese promoters in HCT116 (−∕−) than in HCT116 (þ∕þ) cells,particularly within the mdm2P2 and pcna promoters (2.8-foldand 2.1-fold, respectively). Moreover, the level of nucleosomeoccupancy within the TATA box region of the p21 promoterwas significantly elevated in p53 − ∕− cells (approximately 2.0-fold to 2.3-fold). This indicates an important but poorly under-stood role of p53 in formation of the chromatin landscape withinthe promoters of p53-induced genes that merits further detailedinvestigation.

While this manuscript was in preparation, Lidor et al. pub-lished an interesting study in which, using a custom DNA micro-array, they analyzed the distribution of approximately 2,000 p53binding sites within chromatin in unstressed vs. stressed cells andtheir relative affinities to p53 (31). They made the unexpectedobservation that p53 binding sites reside preferentially withingenomic regions with relatively high intrinsic nucleosome occu-pancy. They showed as well that upon DNA damage nucleosomesare partially and reversibly displaced from a region surroundingbound p53 sites. However, these authors did not directly addresswhether or how p53 binds directly to nucleosomes. Our study,though limited to the p21 p53 REs, has both delved in more detailinto the nucleosomal organization of the p21 distal and proximalREs in vivo and revealed that p53 binds directly to nucleosomalDNA both in vivo and in vitro. Our work has also suggested pos-sible mechanisms by which p53 does so and sets the stage forfuture mechanistic experiments that can reveal in greater detailthe events related to p53 binding to its cognate sites in cells.

MethodsDetails of methods used in this paper including cell culture, protein expres-sion and purification, MNase based experiments, ChIP, ligation-mediatedPCR, and in vitro nucleosome binding experiments are described in SIMaterials and Methods in SI Appendix.

ACKNOWLEDGMENTS. We thank members of the Prives laboratory forcomments and suggestions and are particularly grateful to Dr. David Gross(Louisiana State University) for helpful suggestions and for providing us withthe HSP82 promoter DNA template. This work was supported by NationalInstitutes of Health Grant CA77742.

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