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

Abstract

It is well established that p53 contacts DNA in a sequence-dependent manner in order to transactivate its myriad target genes. Yet little is known about how p53 interacts with its binding site/response element (RE) within such genes in vivo in the context of nucleosomal DNA. In this study we demonstrate that both distal (5') and proximal (3') p53 REs within the promoter of the p21 gene in unstressed HCT116 colon carcinoma cells are localized within a region of relatively high nucleosome occupancy. In the absence of cellular stress, p53 is prebound to both p21 REs within nucleosomal DNA in these cells. Treatment of cells with the DNA-damaging drug doxorubicin or the p53 stabilizing agent Nutlin-3, however, is accompanied by p53-dependent subsequent loss of nucleosomes associated with such p53 REs. We show that in vitro p53 can bind to mononucleosomal DNA containing the distal p21 RE, provided the 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 the p21 gene is located close to the end of the nucleosome. Thus, low- and high-resolution mapping of nucleosome boundaries around p53 REs within the p21 promoter have provided insight into the mechanism of p53 binding to its sites in cells and the consequent changes in nucleosome occupancy at such sites.

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 +11.4 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 (+/+) cells were incubated with 0.75 μM dox followed by processing for ChIP analysis of p53 binding to p21 5′ and 3′ REs and +11.4 kb region as a negative control. (C) HCT116 (+/+) 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.

Fig. 2.

Doxorubincin treatment alters nucleosome distribution near the p21 distal p53 binding site. (A) Schematic representation of the approximately 500-bp region surrounding the p21 5′ p53 RE together with the corresponding amplicons. Each filled segment represents 20 bp and the open segment is the location of the 5′ p53 RE. Note that there is an approximately 60-bp gap between the first and the second amplicons that could not be assayed to due to difficulties with primer design. (B and C) MNase-resistant DNA in the amplified regions shown in A. Mononucleosomal DNA was prepared as described in the SI Materials and Methods in SI Appendix from either HCT116 (+/+) (B) or HCT116 (-/-) (C) cells treated with 0.75 μM doxorubicin for 0 (filled bars) or 8 h (open bars). (D) MNase-assisted ChIP (see SI Materials and Methods in SI Appendix) was used to measure the relative amounts of H3 histone associated with each amplicon in A in HCT116 (+/+) cells. Data are presented as the ratio of histone H3 at 8 hr post Dox/ histone H3 at 0 hr (untreated).

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 (+/+) 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 PhosphorImager 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.

Fig. 3.

p53 is bound to nucleosomal DNA in cells prior to treatment with doxorubicin. MNase-assisted ChIP was performed using HCT116 (+/+) 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 +11.4 region. Dotted line represents the background signal for each experiment.

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. PhosphorImager scans in B were analyzed using ImageQuant Software and presented as graphs (C).