Article Figures & Data
Figures
- FIG. 1.
The p53 gene is organized into two epigenetic domains by DNA methylation characteristics and histone modifications. (A) Schematic representation of the p53 gene showing the position of the promoter and exons as well as the methylation boundaries. The gene is subdivided into 25 regions for PCR analyses of the chromatin structure and ChIP assays. The distribution of CpG methylation was analyzed by both methylation-sensitive PCR and bisulfite sequencing (M, methylated region; Um, unmethylated region; T, upstream methylation transition zone). The positions of CpG sites are shown below the methylation data. (B to E) Distribution of histone modifications in the p53 gene. Specific antibodies used in the ChIP experiments were anti-acetylated H3-K9 (H3K9ac; B), anti-acetylated H4-K16 (H4K16ac; C), anti-dimethylated H3-K4 (H3K4me2; D), and anti-trimethylated H3-K4 (H3K4me3; E). DNA isolated from immunoprecipitated chromatin was subjected to PCR to amplify DNA fragments from the murine p53 locus in the 25 regions specified in panel A. Nonimmune immunoglobulin G (IgG)-immunoprecipitated DNA was used as the control. To determine the relative levels of acetylation and di- and trimethylation in these regions (1 through 25), primers specific for the murine GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene promoter with known histone modification patterns were included in the PCRs as the internal control. The degree of enrichment is calculated relative to the ratio of signals obtained in the input DNA fraction. Data are the means ± standard errors of the means (SEM) from at least three independent experiments.
- FIG. 2.
Unmethylated domain I region of p53 is hypersensitive to MNase digestion and devoid of H2B. (A) PCR analysis of different regions of the p53 gene after PT67 nuclei were digested with increasing amounts of MNase (0, 10, 15, 20, 25, 35, and 75 U/ml) at 37°C for 5 min. After purification, genomic DNA was analyzed by PCR amplification using the primer pairs 1 to 13 as indicated in the p53 map. The unmethylated region is marked and is clearly found to be nuclease hypersensitive. MN conc., MNase concentration. (B) Southern blotting analysis of the nucleosomal organization of epigenetic domains I and II. DNA was extracted from nuclei digested with MNase for 5 min at 37°C with increasing amounts of nuclease (from 10 to 75 U/ml). The DNA was electrophoresed in a 1.2% agarose gel. Left, result of Southern blot analysis using probe A as indicated in the upper portion of the panel. Right, Southern blot analysis of nucleosomal organization of epigenetic domain II using probe B and showing the nucleosomal DNA ladders. conc., concentration. (C) Distribution of H2B of p53 chromatin in the PT67 cells (n = 3; means ± SEM are shown).
- FIG. 3.
Association of several regulatory factors with the p53 epigenetic domains. ChIP assays using anti-CTCF, anti-Topo II, anti-HDAC1, anti-Dnmt1, anti-Brg1, and anti-Topo I antibodies were performed to determine the binding sites of these regulatory factors. Input chromatin and nonspecific IgG precipitate are shown as controls. CTCF, HDAC1, Dnmt1, and Topo II were found in domains I and II, whereas Brg1 and Topo I is found only in domain II. CTCF and Topo II were located in region 13; HDAC1, Brg1, and Topo I were associated with region 12; and Dnmt1 was associated with both region 12 and region 13. α-, anti-.
- FIG. 4.
Depletion of RNA pol II with loss of regulatory factors in the methylated epigenetic domain of p53 gene chromatin in G2/M-arrested cells as analyzed by ChIP. (A) Structure of the p53 gene showing the positions of the promoter, the exons, and the two epigenetic domains. (B and C) Distribution of RNA pol II of p53 in the nonsynchronized cells or in the G2/M-phase cells, respectively. RNA pol II is depleted in the epigenetic domain II in mitotic phase-arrested cells accumulated by treatment with nocodazole (G2/M) compared with the nonsynchronized cells (untreated). (D and E) Loss of regulatory factors from the intragenic region of the p53 gene in mitotic phase-arrested cells. ChIP assays using anti-CTCF, anti-Topo II, anti-PARP1, anti-Brg1, and anti-Topo I antibodies were performed to determine the binding sites of these regulatory factors. α-, anti-.
- FIG. 5.
Inhibition of PARP1 and Topos resulted in the depletion of RNA pol II in the second epigenetic domain and the loss of regulatory factors. (A) Structure of the p53 gene showing the positions of the promoter, the exons, and the two epigenetic domains. (B) Depletion of RNA pol II in domain II in the p53 gene in 3-AB-treated PT67 cells. (C) Treatment with the Topo I inhibitor camptothecin for 30 min resulted in depletion of pol II in domain II of p53. (D) Treatment with the Topo I inhibitor camptothecin for 2 h resulted in an accumulation of pol II in region 12, where Topo I is located (Fig. 3). (E) Treatment with the Topo II inhibitor etoposide for 30 min in vivo resulted in the depletion of pol II in domain I of p53 and the 3′ half of domain II. (F) Treatment with the Topo II inhibitor etoposide for 2 h resulted in the depletion of pol II in the entire gene (G and H). After 3-AB treatment, loss of CTCF, Topo II, PARP1, Brg1, and Topo I from p53 chromatin occurred. Input chromatin and nonspecific IgG precipitate are shown as controls. α-, anti-.
- FIG. 6.
Histone H1 levels appear enriched on epigenetic domain II of p53 chromatin in G2/M-arrested cells as analyzed by ChIP. Distribution of histone H1 of p53 in the nonsynchronized cells or in the G2/M-phase cells, respectively (n = 3; means ± SEM are shown).
- FIG. 7.
Epigenetic domain II sequences including the distal 3′ end interact with the p53-proximal promoter region. (A) Schematic of the p53 gene with the orientation of primers (arrows) and their distances to NcoI sites in the p53 locus are shown. (B) 3C assay shows the presence of the specific 3C products (1-2, 1-3, 1-4, and 1-5) in different drug-treated PT67 cells (G2/M, nocodazole-treated PT67 cells; 3-AB, 3-AB-treated PT67 cells; CPT, camptothecin-treated PT67 cells; G1/S, aphidicolin-treated PT67 cells) under conditions in which formaldehyde (CH2O) cross-linking and ligation of the samples were carried out. The loading control primers (gapdh promoter) showed that all of the samples contained similar amounts of DNA.
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Fig. S1 (Partial methylation patterns in the upstream methylation boundary).
Zipped MS Word document, 254K. - Supplemental file 2
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Fig. S2 (Three nuclease hypersensitive sites in the p53 promoter region).
Zipped MS Word document, 214K. - Supplemental file 3
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Fig. S3 (p53 CTCF-binding sequence functional assay).
Zipped MS Word document, 153K. - Supplemental file 4
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Fig. S4 (Distribution of H3S10P in p53).
Zipped MS Word document, 108K. - Supplemental file 5
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Table S1 (Primers for bisulfite methylation assays).
MS Word document, 82K. - Supplemental file 6
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Table S2 (Oligonucleotides for EMSA and enhancer-blocking assay).
MS Word document, 28K. - Supplemental file 7
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Table S3 (Primers for ChIP and MNase reaction).
MS Word document, 51K. - Supplemental file 8
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Fig. S5 (Run-on assay of p53 transcription).
Zipped MS Word document, 174K. - Supplemental file 9
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Fig. S6 (Distribution of H2B in p53).
Zipped MS Word document, 46K. - Supplemental file 10
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Supplemental text.
Zipped MS Word document, 24K.
- Supplemental file 1
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Fig. S1 (Partial methylation patterns in the upstream methylation boundary).
