P53 Binding to Nucleosomes Within the P21 Promoter in Vivo Leads To

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p53 binding to nucleosomes within the p21 INAUGURAL ARTICLE promoter in vivo leads to nucleosome loss and transcriptional activation

Oleg 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- but can be separated by up to 13 bp (9–11). Multiple studies in dependent manner in order to transactivate its myriad target recent years have focused on the interaction of p53 with its genes. Yet little is known about how p53 interacts with its binding cognate binding sites in vivo and in vitro and subsequent gene site/response element (RE) within such genes in vivo in the context transactivation (or transrepression). Here we have examined the of nucleosomal DNA. In this study we demonstrate that both distal nucleosomal status in vivo of p53 binding sites within one of its (5′) and proximal (3′) p53 REs within the promoter of the p21 gene major target genes, p21, before and after induction of p53 and in unstressed HCT116 colon carcinoma cells are localized within a have also determined the extent to which p53 is able to interact region of relatively high nucleosome occupancy. In the absence of with its cognate sites within nucleosomal context. cellular stress, p53 is prebound to both p21 REs within nucleosomal DNA in these cells. Treatment of cells with the DNA-damaging drug Results doxorubicin or the p53 stabilizing agent Nutlin-3, however, is p53-Dependent Loss of Nucleosomes Occurs at p53 Binding Sites accompanied by p53-dependent subsequent loss of nucleosomes Within the p21 Promoter. We focused on p21 as it is one of the best associated with such p53 REs. We show that in vitro p53 can bind characterized bona fide p53 target genes. The p21 promoter BIOCHEMISTRY to mononucleosomal DNA containing the distal p21 RE, provided has two p53 binding sites (or response elements, REs) that con- the binding site is not close to the diad center of the nucleosome. form to the p53 consensus binding sequence (Fig. 1A), the more In line with this, our data indicate that the p53 distal RE within the distal (5′) site at −2283 that binds p53 relatively strongly and the p21 gene is located close to the end of the nucleosome. Thus, low- more proximal (3′) site at −1391 that is more weakly bound by and high-resolution mapping of nucleosome boundaries around p53 (12–14). We first determined the nucleosome status at these p53 REs within the p21 promoter have provided insight into the sites between matched HCT116 colon carcinoma cell lines that mechanism of p53 binding to its sites in cells and the consequent either contain (þ∕þ) or lack (−∕−) full-length p53 (15). As ex- changes in nucleosome occupancy at such sites. pected, treatment with doxorubicin (dox) resulted in a significant increase of p53 levels in HCT116 (þ∕þ) cells and this preceded DNA binding ∣ DNA damage increases in both p21 mRNA and protein (Fig. S1 in SI Appendix). Activation of p21 expression correlated with p53 binding to its 5′ ′ n eukaryotic cells, genomic DNA is tightly associated with his- and 3 REs as measured by chromatin immunoprecipitation B tones resulting in the organized and dynamic structure known as (ChIP) (Fig. 1 ). Consistent with the known higher affinity of I ′ ′ chromatin (1, 2). The primary unit of chromatin is the nucleo- p53 for the 5 RE than for the 3 RE, there was significant basal some, which is composed of approximately 146 bp DNA wound binding to the former site that was even equivalent to binding to ′ around the core histone octamer (3). The resulting higher- the 3 site after 4 h of dox treatment. To examine the nucleosome p21 ordered structure helps to compact the DNA within the nucleus. status of the p53 REs within the promoter, we determined the At the same time it represents an accessibility barrier for specific extent to which these regions were resistant to micrococcal nu- transcription factors whose primary role is to regulate the multi- clease (MNase) digestion as reported for other transcribed genes step process of transcriptional activation of most gene promoters (16). Cells were treated or not with dox followed by cross-linking in response to pathway-activating stimuli. Many in vitro biochem- with formaldehyde (to preserve chromatin structure) and then ical studies have revealed significant reduction in transcription nuclei were isolated and incubated with MNase. DNA recovered factor binding affinities toward their cognate sites within nucleo- from the MNase-resistant mononucleosomal fraction was somal DNA as compared to naked DNA. In vivo, a number of extracted from the gel and subjected to Q-RT-PCR analysis to assess the nucleosome status in the vicinity of the two p53 molecular mechanisms may promote specific and efficient inter- p21 actions between a given transcriptional regulator and its binding REs in the promoter region as well as in two control regions: the TATA box at −20 bp and further downstream þ11.4 kb. In site within DNA. Some of these mechanisms depend on the þ∕þ −∕− enzymatic activities of chromatin remodeling complexes that fa- unstressed HCT116 ( )or( ) cells both p53 REs had a cilitate either nucleosome eviction or sliding (4), while others rely relatively high nucleosome content when compared to the TATA region that was previously reported to be bound by RNA poly- on cooperative binding between transcription factors (5), local C “ ” histone modifications (6), and/or prior nucleosome interactions merase II and virtually nucleosome free (17) (Fig. 1 , time 0 ). “ ” Notably, dox treatment resulted in a rapid p53-dependent loss of with so-called pioneer factors (7). C D p53 is a sequence-specific transcriptional activator that exerts nucleosomal content within both p53 REs (Fig. 1 and ), while its tumor-suppressor activity primarily through regulation of transcription initiation of multiple downstream target genes (8). Author contributions: O.L. and C.P. designed research; O.L., R.B., and E.F. performed Mutations within the DNA-binding domain (DBD) of p53 and research; O.L., R.B., and C.P. analyzed data; and O.L. and C.P. wrote the paper. subsequent loss of specific DNA-binding activity are responsible The authors declare no conflict of interest. for p53 inactivation in more than 50% of tumors. The p53 con- 1To whom correspondence may be addressed. E-mail: [email protected]. sensus binding site is quite complex and consists of two decameric This article contains supporting information online at www.pnas.org/lookup/suppl/ half-sites, RRRCA/TT/AGYYY that are usually directly adjacent doi:10.1073/pnas.1105680108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1105680108 PNAS ∣ June 28, 2011 ∣ vol. 108 ∣ no. 26 ∣ 10385–10390 Downloaded by guest on September 25, 2021 Fig. 1. Doxorubicin treatment leads to p53-dependent loss of nucleosomes at p53 binding sites within the p21 promoter. (A) p21 promoter region showing p53 5′ and 3′ REs, TATA region and þ11.4 kb control region. Bold arrow represents the p21 transcription start site and head-to-head oriented pairs of arrows represent the sites that were assayed for MNase hypersensitivity by Q-RT-PCR amplification. (B) HCT116 (þ∕þ) cells were incubated with 0.75 μM dox followed by processing for ChIP analysis of p53 binding to p21 5′ and 3′ REs and þ11.4 kb region as a negative control. (C) HCT116 (þ∕þ) cells were treated with 0.75 μM dox for indicated time periods as above, followed by formaldehyde cross-linking and processing to determine relative mononucleosomal occupancy at the p21 distal 5′ and proximal 3′ p53 REs as well as the p21 TATA region. MNase-digested chromatin were deproteinized and mononucleosomal DNA was gel-purified for use as templates in Q-RT-PCR with pairs of primers flanking indicated regions within the p21 promoter. (D) Same as in C performed using HCT116 p53 (−∕−) cells. (E) MNase hypersensitivity data obtained for two control regions within p21 gene, with relatively high (11.4 kb site), and low (TATA) initial nucleosomal content.

we detected no p53-associated increase in MNase sensitivity with- SI Appendix). For analysis of those, we employed the ligation- in any of the two control regions (Fig. 1E). mediated (LM) PCR technique (see Fig. 5 below). Because dox can induce DNA strand breaks and thereby acti- Unexpectedly, this low-resolution mapping experiment re- vate a number of intracellular processes associated with chroma- vealed the presence of two distinct (though closely spaced) sites tin remodeling independently of p53 (18), we performed the of MNase sensitivity within the distal p53 RE locus in HCT116 same experiment using Nutlin-3 that disrupts the p53 interaction þ∕þ) cells (Fig. 2 B–D). The first coincides with the original p53 with Mdm2 and thereby stabilizes p53 in the absence of DNA RE itself (primer pairs 2 and 3) and the second site is located damage (19). Nutlin-3 induced p21 RNA accumulation that about 150–160 bp 3′ to the RE (primer pair 5). These alterations was accompanied by a decrease in MNase-resistant DNA at both required full-length p53 because there were no significant p53 REs (Fig. S2 A–C in SI Appendix). The kinetics of nucleo- changes within the corresponding regions in HCT116 −∕− cells C þ∕þ some loss from both p53 REs upon treatment with either p53 (Fig. 2 ). In HCT116 ( ) cells the relative increases in MNase stabilizing agent was very similar. Note that dox-induced loss of sensitivity upon treatment with dox were accompanied by a nucleosomes within the vicinity of the 5′ RE in HCT116 (þ∕þ) significant loss of histones within both regions as shown by D cells required the presence of p53, because introduction of p53 an MNase-assisted ChIP experiment (Fig. 2 ). Interestingly, siRNA but not control siRNA into these cells prior to drug treat- between these two hypersensitive regions we also detected a site ment resulted in significant loss of MNase hypersensitivity within that became more resistant to MNase under conditions that that region (Fig. S3 in SI Appendix). activate p53, and MNase-assisted ChIP analysis of that region We asked whether the altered nucleosome occupancy at the revealed that it, too, had a relative increase in local histone p53 REs depended upon ongoing transcription of p21 RNA by occupancy (primer pair 4). Although p53 binds far more weakly to the proximal RE in the performing a similar experiment to the one shown in Fig. 1 but p21 promoter, the MNase sensitivity profile within that region in the presence or absence of the potent RNA Polymerase II in- α SI Appendix was very similar to that seen at the stronger distal RE element hibitor, -amanitin (Fig. S4 in ). While transcription B B SI Appendix p21 puma mdm2 (compare the data from Fig. 2 with Fig. S5 of ). of several p53-dependent targets such as , , and This region showed two MNase-sensitive sites (primer pairs 2, was blocked by α-amanitin regardless of p53 activation status A SI Appendix 3, and 6) separated by a region where there was no significant (Fig. S4 in ), MNase hypersensitivity within the change in MNase-resistant DNA (primer pairs 4∕5). There were p21 B SI REs remained unchanged by the inhibitor (Fig. S4 in also corresponding changes in histone H3 occupancy within this Appendix ). Thus, p53 does not require active transcription to region. As with the 5′RE region, there were only very modest occur in order for it to initiate eviction of nucleosomes within the dox-dependent changes in nucleosome distribution at the regions of its binding sites. p21 proximal RE in the absence of full-length p53 (Fig. S5C in SI Appendix). Localized p53-Dependent Eviction of Nucleosomes Occurs at the p53 REs Within the p21 Promoter. We extended our analysis of the nu- p53 Is Bound to Nucleosomal DNA in Unstressed HCT116 Cells. cleosome density changes within chromatin at the p53 REs of Although the above mapping experiments elucidated features p21 the promoter after dox treatment. For this purpose, multiple of the chromatin organization within the distal and proximal primer pairs were designed covering 450–500 bp surrounding p53 REs, they did not reveal whether p53 can bind directly to either the distal or proximal p53 REs (Fig. 2A and Fig. S5A in mononucleosomal DNA in vivo. To address this mononucleo- SI Appendix, respectively). The length of an average amplicon somes prepared from formaldehyde-crosslinked chromatin by obtained in a Q-PCR reaction with either primer pair was limited digestion with MNase were immunoprecipitated with approximately 85 bp and sequences of neighboring amplicons either anti-p53 or anti-H3 antibodies. The recovered mononu- overlapped by approximately 10 bp. Unfortunately, we could cleosomal DNA was amplified by Q-PCR using primer pairs spe- not design primers that would cover approximately 60 bp gaps cific to the p21 p53 distal and proximal REs, or the two negative that were 5′ to either the distal or the proximal site (between the control regions at the TATA and þ11443 regions (Fig. 3). The first and second primer pairs; see Fig. 2A and Fig. S5A in results strongly indicate that in unstressed cells p53 is bound

10386 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105680108 Laptenko et al. Downloaded by guest on September 25, 2021 markedly greater than those with either the TATA or þ11443 regions where there was also very little change after dox treatment. Note that while the amount of specific mononucleosomal DNA INAUGURAL ARTICLE immunoprecipitated with the p53 antibodies was reduced upon dox-dependent p53 activation (as measured by MNase-assisted ChiP), total p53 binding to the p21 REs was increased over the same time period after dox treatment (compare the data from Fig. 3 and from Fig. 1B). Thus, loss of the signal upon p53 activation in the MNase-assisted ChiP assay simply reflects the loss of nucleosomes but not the loss of overall p53 DNA-binding per se.

Stable Nucleosomal Binding by p53 Requires That Its Site Be Close to the End of Nucleosomal DNA. With few exceptions biochemical studies on p53/DNA interactions have been examined in the context of naked DNA. We therefore sought to recapitulate our ob- servations showing that p53 binds to its sites within chromatin at the p21 promoter by examining how p53 interacts with mononucleosomes in vitro. We performed DNase I footprinting using either naked or reconstituted mononucleosomal DNA spanning the p21 promoter p53 REs (Fig. S6 in SI Appendix). A strong nucleosome positioning sequence derived from the yeast HSP82 promoter region was incorporated into a chimeric 170 bp DNA construct that had the p21 distal p53 RE located 30 bp from the 5′-end (see SI Materials and Methods in SI Appendix for details). As expected, preincubation of naked DNA with increasing amounts of purified wild-type p53 protein resulted in strong pro- Fig. 2. Doxorubincin treatment alters nucleosome distribution near the p21 tection from DNase I cleavage within the RE region (Fig. S6A in distal p53 binding site. (A) Schematic representation of the approximately SI Appendix). As described previously (20), when the same DNA BIOCHEMISTRY 500-bp region surrounding the p21 5′ p53 RE together with the correspond- was present within a mononucleosome, DNase I cleavage yielded ing amplicons. Each filled segment represents 20 bp and the open segment a characteristic approximately 10-bp periodicity pattern that ′ is the location of the 5 p53 RE. Note that there is an approximately 60-bp revealed the contacts between the DNA and the core histone oc- gap between the first and the second amplicons that could not be assayed to tamer (Fig. S6B in SI Appendix). p53 binding to this mononucleo- due to difficulties with primer design. (B and C) MNase-resistant DNA in the amplified regions shown in A. Mononucleosomal DNA was prepared as somal DNA resulted in clear but relatively weak protection of the described in the SI Materials and Methods in SI Appendix from either p21 gene distal RE (see densitometry analysis) along with an HCT116 (þ∕þ)(B) or HCT116 (−∕−)(C) cells treated with 0.75 μM doxorubicin appearance of a DNase I hyper-sensitive site located at the 3′ for 0 (filled bars) or 8 h (open bars). (D) MNase-assisted ChIP (see SI Materials edge of the RE sequence (indicated by asterisk). and Methods in SI Appendix) was used to measure the relative amounts of An electrophoretic mobility shift assay (EMSA) was also per- H3 histone associated with each amplicon in A in HCT116 (þ∕þ) cells. Data formed to characterize p53 interactions with mononucleosomal are presented as the ratio of histone H3 at 8 hr post Dox/ histone H3 at 0 hr DNA in vitro (Fig. 4). Our initial experiments revealed that (untreated). p53 did not detectably bind to such DNA from the p21 promoter when the p53 5′ RE was centrally positioned (Fig. S7 in SI to MNase-resistant DNA and, depending on the site, such bind- Appendix) so we assessed whether p53 might bind to its site at ing is either greatly decreased or completely lost upon treatment other positions within mononucleosomal DNA. To this end, we with dox, which is mirrored by significant loss of histones within prepared a set of ten 170-bp chimeric DNA templates containing the p53 REs. Supporting this likelihood, the signal at the REs was the p21 p53 5′ RE located at positions that differed by one base

Fig. 3. p53 is bound to nucleosomal DNA in cells prior to treatment with doxorubicin. MNase-assisted ChIP was performed using HCT116 (þ∕þ) cells treated with 0.75 μM doxorubicin for 0 (filled bars) or 8 (open bars) hours. Q-RT-PCR was used to measure MNase-resistant DNA immunoprecipitated with either anti-p53 or anti-H3 antibodies as indicated at (A) p21 5′ p53 RE, (B) p21 3′ p53 RE, (C) p21 TATA region, or (D) p21 þ11.4 region. Dotted line represents the background signal for each experiment.

Laptenko et al. PNAS ∣ June 28, 2011 ∣ vol. 108 ∣ no. 26 ∣ 10387 Downloaded by guest on September 25, 2021 Fig. 4. Detectable p53 binding to the p21 distal RE requires the site to be positioned close to the end of a mononucleosome. (A) The location of p21 distal p53 REs inserted within 170 BP DNA fragments from yeast HSP82 sequence containing a strong positioning sequence assembled into mononucleosomal DNA by the octamer transfer method. (B) An electrophoretic mobility shift assay was performed with [32P] labeled mononuclesomal DNAs shown in A and purified p53 protein. Four different DNA/mononucleosomes were run on one gel, and then construct (170–30) was re-run on each gel to correct differences between the experiments. Shown are PhosphorImager scans of three 4% native 0.5X TBE gels with 10 mononucleosomal constructs. Phosphor- Imager scans in B were analyzed using ImageQuant Software and presented as graphs (C).

pair in their distance from the 5′-end of the DNA (Fig. 4A). (the most prominent one situated about 55 bp away from the RE) EMSA analysis showed that p53 binding to mononucleosomes that again likely represent multiple potential nucleosome bound- became detectable and increased as the RE was moved further aries (Fig. S8 in SI Appendix). Here too there was a significant from the center of the DNA. In fact, a 2-bp shift in the RE loss of intensity of the major band/boundary and almost total (170–30) resulted in an approximately fivefold increase in binding vanishing of minor bands 8 h after administration of dox. to mononucleosomal DNA when compared to a mononucleosome where the RE was located 32 bp from the end of the DNA Discussion p21 (170–32), and a 9-bp shift in the RE toward the center (170–39) was one of the first genes found to be positively regulated by reduced binding by more than a factor of 10 (see Fig. 4 B and C, p53 (12, 22). Although a number of studies have addressed the binding of p53 to its distal and proximal binding sites within the graph). Thus, the distance between the binding site and the end of p21 the DNA represents a critical parameter influencing binding of promoter in vitro and in vivo, none has interrogated such binding in the context of mononucleosomal DNA in detail. In this p53 to mononucleosomal DNA. A similar dependence of binding study we have gained insight into chromatin organization in the to the site within nucleosomal DNA was demonstrated before for p21 promoter, in particular within regions spanning its two p53 other sequence-specific proteins (e.g., for restriction endonu- REs. By applying ChIP, MNase hypersensitivity assays, MNase- cleases) (21). assisted ChIP, and LM PCR, we demonstrate that p53 REs within the p21 promoter in unstressed HCT116 cells are localized within Mapping the Location(s) of the Distal p53 Binding Site Within a Nucleo- nucleosomal DNA. Further, nucleosomes are rapidly lost upon some in the p21 Promoter in Vivo. We next sought to determine the activation of p53 with either the DNA-damaging agent doxoru- location of the p21 distal p53 binding site within the relevant bicin or by blocking its degradation by Mdm2 through adminis- mononuclesome in vivo by performing linker-mediated PCR 116 þ ∕þ tration of Nutlin-3. Changes in hypersensitivity within both p53 (LMPCR) in HCT cells (Fig. 5). This analysis revealed p21 – REs of promoter may be explained by either nucleosome several major primer extension products (numbered 1 5) located sliding or nucleosome eviction. After p53 activation by dox, with- at different distances from the p53 RE that we interpret as repre- in the vicinity of the p21 5’ RE there is an apparent loss of two senting nucleosomal boundaries. In Fig. 5 products numbered 1 ′ nucleosomes (Fig. 2, amplicons 2 and 5) and between them there and 2, that are 107 and 68 bp from the p53 5 RE, respectively, is a relative increase in H3 occupancy within amplicon 4. Yet the likely represent the boundaries of two neighboring nucleosomes. distance covered by amplicon 4 is not long enough to contain a Additionally, three products of weaker intensity (numbered 3, 4, full nucleosome core particle (i.e., consisting of the cannonical and 5) were situated much closer to the p53 RE at 41, 32, and histone octamer). Conceivably partial loss of a portion of the his- 18 bp, respectively. Indeed, consistent with our in vitro binding tones from that nucleosome and/or a subsequent clash between data, the location of these products positions the p53 binding site two neighboring nucleosomes has occurred. This is not unheard close to the edge of the nucleosome, thus making it more acces- of—see, for example, Dechassa et al. (23). Using in vitro recon- sible for p53. Further, all above-mentioned species were reduced stituted mono-, di-, and three-nucleosome arrays in the presence upon dox treatment, which is in good agreement with the data of SWI/SNF, those authors demonstrated collision of two neigh- obtained from the low-resolution mapping experiment shown boring nucleosomes, as well as partial loss of H2A/H2B dimer. As in Fig. 2B. This pattern of LMPCR extension products suggests discussed below, the presence of certain histone variants (H3.3 that the nucleosome at the distal p53 RE is somewhat heteroge- and H2A.Z, in particular) and their effect on nucleosome stability neously positioned. We assume that formaldehyde cross-linking may also be in play here. prior to MNase treatment helped us to catch a nucleosome that Does p53 bind to nucleosomal DNA and cause loss of nucleo- contains the p53 distal RE in unstressed cells in several most- somes (directly by destabilizing multiple histone-DNA contacts, probable positions (i.e., positions 2, 3, 4, and 5). Analysis of or indirectly by bringing down components of chromatin remo- the proximal 3’ p53 RE by LMPCR also revealed multiple species deling machinery), or does nucleosome loss precede and is neces-

10388 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105680108 Laptenko et al. Downloaded by guest on September 25, 2021 INAUGURAL ARTICLE

Fig. 5. High-resolution mapping of MNase-sensitive sites indicate that the p21 distal p53 binding site is close to the end of a nucleosome in vivo. HCT116 (þ∕þ) p53 cells were treated with 0.75 μM doxorubicin for 0 or 8 hours after which cells were cross-linked with formaldehyde followed by isolation of nuclei and treatment with three different concentrations of MNase. Deproteinized, purified DNA was subjected to LM PCR as described in SI Materials and Methods in SI Appendix. Left panel shows PhosphorI- mager scan of the 9% polyacrylamide gel that resolved the LMPCR products. AGCT lanes are DNA markers obtained in primer extension reactions with acyclo-ATP, -GTP, -CTP, and -TTP, respectively (New England Biolabs). Graph on right shows densitometry analysis of the gel products at 0 and 8 hours after dox treatment. Putative nucleosome boundaries deduced from MNase-sensitive sites and their ′

locations relative to the p53 5 RE are depicted below. BIOCHEMISTRY

sary for p53 sequence-specific binding? The necessity of nucleo- as positioning of the RE in relatively close proximity to the end of some removal for gene activation was suggested more than two the nucleosome. Indeed, our LM PCR and EMSA experiments decades ago. Since then, numerous in vitro binding experiments support this theory. Interestingly, large scale genome analysis of performed on reconstituted nucleosomal substrates have almost data on the distribution of nucleosomes within the p21 promoter exclusively supported the idea that efficient binding of a given in either A375 or MDA-kb2 cell lines indicates that both p53 transcription factor to its RE is greatly reduced in the context REs are situated in regions with fading nucleosomal density, very of a nucleosome. Factors that were shown to interact with nucleo- close to a relatively nucleosome-depleted part of the promoter somes (e.g., TFIIIA, Sp1) have significantly reduced binding (UCSC Genome browser on Human, March 2006 Assembly; affinities when compared to naked DNA (5). Given the complex- NCBI36/hg18). Our low- and high-resolution mapping experi- ity of p53 REs and multiplicity of contacts between the p53 ments correlate well with these findings. We acknowledge that core domain and DNA sequences within each RE (24, 25), we nucleosomal profiles differ between cell lines, which could be assumed that p53 would not bind detectably to its sites when related to changes in DNA such as mutations, deletions, or am- wrapped around the histone octamer. Surprisingly, our MNase- plifications, as well as epigenetic changes, all of which may con- p21 assisted ChIP experiment performed on distal and proximal tribute to the chromatin landscape within a given cell, tissue or REs demonstrated that p53 in unstressed cells is bound to nucleo- organism. somal DNA. What is the significance of such binding when In addition to showing that p53 is able to bind to nucleosomal p53 becomes activated by stress signals? We envision two possible DNA both in vitro and in vivo, our study raises the possibility of a scenarios. In the first, increasing amounts of stabilized p53 tran- potential second p53 binding site that would reside approximately siently compete with a nucleosome for the RE. In this case, when 150–160 bp downstream of the bona fide distal p21 RE (Fig. 2, p53 levels are increased (e.g., after DNA damage) the relatively primer pair 5). This is supported by increased p53-dependent low affinity of p53 for its binding site within the nucleosome might MNase sensitivity and p53-dependent loss of core histones as still be enough to result in efficient transcriptional activation in vivo. Indeed, recent experiments using fluorescence recovery well as the presence of three overlapping weak p53 half-sites after photobleaching (FRAP) have revealed that the nature of within that region. Whether the putative second binding region transcription factor (TF) binding to chromatin is very transient, exists and is functional in vivo remains to be answered by future and the time that a given TF spends on its RE in vivo may be experiments. less than a minute (reviewed in ref. 26). This time frame may be Local histone modifications and/or histone variants may also sufficient to bring down the components of modifying and/or affect p53 binding to nucleosomes. For example, the histone H2 p21 remodeling machinery leading to local nucleosome eviction/dis- variant, H2A.Z, is enriched at p53 REs within the promoter placement, thereby culminating in formation of a competent pre- and DNA-damage stress caused by doxorubicin leads to eviction initiation complex and subsequent activation of transcription. of H2A.Z in a p53-dependent but transcription-independent Experiments that employ FRAP and/or single molecule technol- manner (28). The presence of H2A.Z may positively influence ogy may provide estimates of the half-life of p53 bound to its REs p53 binding to mononucleosomes because the H2A.Z/H2B within nucleosomes or chromatin. histone dimer is less stable than the regular H2A/H2B dimer The second scenario depends on the ability of nucleosomes to and can be relatively easily released from nucleosomes (29). adopt multiple positions within certain regions, which is dictated Experiments involving in vitro p53 binding to the RE localized both by the DNA sequence and components of the chromatin within mononucleosome reconstituted with either H2A or remodeling machinery (reviewed in ref. 27). In this case, initial H2A.Z may provide insight into the relative affinities of such sub- p53 binding will occur under the most favorable conditions, such strates toward p53.

Laptenko et al. PNAS ∣ June 28, 2011 ∣ vol. 108 ∣ no. 26 ∣ 10389 Downloaded by guest on September 25, 2021 A different mechanism for p53-nucleosome interaction was While this manuscript was in preparation, Lidor et al. pub- proposed by Sahu et al. based on the results of an elegant in vitro lished an interesting study in which, using a custom DNA micro- study (30). The authors suggest that orientation of the p53 bind- array, they analyzed the distribution of approximately 2,000 p53 ing site on a nucleosome in conjuction with a nucleosomal posi- binding sites within chromatin in unstressed vs. stressed cells and tioning sequence may preset the p53 RE in a way that is easily their relative affinities to p53 (31). They made the unexpected accessible to p53. Though attractive, confirmation of this model observation that p53 binding sites reside preferentially within requires support from experiments on p53 binding to bona fide REs in the context of their natural settings in vivo. genomic regions with relatively high intrinsic nucleosome occu- Certain p53 REs may localize within nucleosome-depleted re- pancy. They showed as well that upon DNA damage nucleosomes gions. In these cases p53 binding may be a different and less com- are partially and reversibly displaced from a region surrounding plex process and could be regulated by different mechanism(s) bound p53 sites. However, these authors did not directly address such as relative strength of the RE, local concentrations of p53, whether or how p53 binds directly to nucleosomes. Our study, modification status of p53, or cooperative binding with other though limited to the p21 p53 REs, has both delved in more detail transcription factors. Indeed, the results of MNase hypersensitiv- into the nucleosomal organization of the p21 distal and proximal ity analysis performed on several p53-dependent promoters in REs in vivo and revealed that p53 binds directly to nucleosomal þ∕þ −∕− HCT 116 and cells rather support this assumption DNA both in vivo and in vitro. Our work has also suggested pos- (Fig. S9 in SI Appendix). The levels of basal nucleosome occu- sible mechanisms by which p53 does so and sets the stage for pancy differ drastically at p53 REs within the mdm2P2, bax, puma,orPCNA promoters: Whereas the first two are virtually future mechanistic experiments that can reveal in greater detail nucleosome free, bax and (even more so) puma p53 REs show the events related to p53 binding to its cognate sites in cells. strong MNase resistance. Interestingly as well, there is relatively Methods higher basal nucleosome occupancy detected within p53 REs of −∕− þ∕þ Details of methods used in this paper including cell culture, protein expres- these promoters in HCT116 ( ) than in HCT116 ( ) cells, sion and purification, MNase based experiments, ChIP, ligation-mediated particularly within the mdm2P2 and pcna promoters (2.8-fold PCR, and in vitro nucleosome binding experiments are described in SI and 2.1-fold, respectively). Moreover, the level of nucleosome Materials and Methods in SI Appendix. occupancy within the TATA box region of the p21 promoter was significantly elevated in p53 −∕− cells (approximately 2.0- ACKNOWLEDGMENTS. We thank members of the Prives laboratory for fold to 2.3-fold). This indicates an important but poorly under- comments and suggestions and are particularly grateful to Dr. David Gross stood role of p53 in formation of the chromatin landscape within (Louisiana State University) for helpful suggestions and for providing us with the promoters of p53-induced genes that merits further detailed the HSP82 promoter DNA template. This work was supported by National investigation. Institutes of Health Grant CA77742.

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