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

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P53 Binding to Nucleosomes Within the P21 Promoter in Vivo Leads To 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-depen- dent 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 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 binding to p21 5′ and 3′ REs and þ11.4 kb region as a nega- tive control. (C) HCT116 (þ∕þ) cells were treated with 0.75 μM dox for indicated time periods as above, fol- lowed 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 with- in the p21 promoter. (D) Same as in C performed using HCT116 p53 (−∕−) cells. (E) MNase hypersensi- tivity 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.
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