Article Figures & Data
Figures
- Fig. 1.
Map of histone H3 modifications along a hypermethylated versus an unmethylated hMLH1 promoter. A, schematic of the hMLH1 promoter. The vertical lines represent the location of CpG dinucleotides, and the arrow indicates the approximate position of the transcription start site. The CpG island extends 3′ from ∼−800 (relative to the transcription start site) into exon 1. The doubled horizontal line denotes the region examined by MSP. In SW480 cells, the promoter is unmethylated, and the gene is expressed. However in RKO cells, the promoter is hypermethylated, and the gene is silenced. The horizontal bars below the schematic indicate the location of the DNA fragments amplified by PCR done on the DNA recovered from ChIP experiments. The broken bars denote the primer sets used in the time course experiments. B, D, and F, enrichment of hMLH1 promoter DNA immunoprecipitated with antibodies specific for acetylated histone H3 (K9 and K14), dimethyl-H3-K4, and dimethyl-H3-K9, respectively. Points on the graphs represent data from the corresponding DNA fragment amplified by PCR, as shown at the bottom A. The value of each point was calculated as the average from two independent ChIP experiments and a total of four independent PCR analyses. Each error bar indicates the SD from the mean. □ represent data from SW480. ▪ represent data from RKO. C, E, and G, representative PCR analyses of ChIP on RKO and SW480 from areas typical of enrichment for acetylated H3, methyl-H3-K4, and methyl-H3-K9, respectively. Multiplex PCR was performed on bound (B) immunoprecipitated DNA and input (I) nonimmunoprecipitated DNA with each hMLH1 primer set.
- Fig. 2.
Inhibition of histone deacetylation by TSA fails to dramatically alter key components of the histone code map along the hypermethylated hMLH1 promoter. ChIP was done on RKO cells after treatment with 300 nm TSA for 24 h. A, C, and E, enrichment of acetylated histone H3 (K9 and K14), dimethyl-H3-K4, and dimethyl-H3-K9, respectively, at the hMLH1 promoter. ○ represents enrichment in RKO cells treated with TSA. • represents data from untreated RKO cells. Points on each graph represent data from the corresponding DNA fragment amplified by PCR, as illustrated in Fig. 1A<$REFLINK> . The value of each point was calculated as the average from two independent ChIP experiments and a total of four independent PCR analyses. Each error bar indicates the SD from the mean. B, D, and F, representative PCR analyses of ChIP performed on RKO cells, before and after treatment with TSA, from areas typical of enrichment for acetylated H3, methyl-H3-K4, and methyl-H3-K9, respectively. Bound DNA (B) and input DNA (I) were coamplified with primers for hMLH1 and GAPDH.
- Fig. 3.
Inhibition of DNA methylation by 5-Aza-dC completely reverses all examined components of the histone code map along the hypermethylated hMLH1 promoter. ChIP was performed on RKO cells after treatment with 1 μm 5-Aza-dC for 5 days. A, C, and E, enrichment of hMLH1 promoter DNA precipitated by antibodies specific for acetylated histone H3 (K9 and K14), dimethyl-H3-K4, and dimethyl-H3-K9, respectively. ○ represents enrichment in RKO cells treated with 5-Aza-dC. • represents data from untreated RKO cells. The value of each point was calculated as the average from two (untreated) or three (drug-treated) independent ChIP experiments and four independent PCR analyses from each untreated or drug-treated experiment. Each error bar indicates the SD from the mean. Points on each graph correspond to the overlapping DNA fragments amplified by PCR as depicted in Fig. 1A. B, D, and F<$REFLINK> , representative PCR analyses of ChIP done on RKO cells, with or without treatment with 5-Aza-dC, from areas typical of enrichment for acetylated H3, methyl-H3-K4, and methyl-H3-K9, respectively. DNA from bound (B) and input (I) fractions were coamplified with primers for hMLH1 and GAPDH.
- Fig. 4.
Treatment with 5-Aza-dC reverses all components of the histone code examined at a hypermethylated hMLH1 promoter by 48 h. The data represent two independent time course experiments in which RKO cells were treated with 1 μm 5-Aza-dC (or mock-treated) and harvested at each time point shown for ChIP analysis. A total of four to seven PCR analyses were performed on the immunoprecipitated DNA from each time point. Each point on the graphs represents the average value of enrichment, and each error bar indicates the SD from the mean. ○ represents data from RKO cells treated with 5-Aza-dC. • represents data from mock-treated RKO cells. Data from a 5-day time point served as positive controls to ensure that drug treatment was effective. The broken horizontal bars under the hMLH1 promoter schematic in Fig. 1A<$REFLINK> indicate the location of the primer sets used in this ChIP and PCR analysis.
- Fig. 5.
Treatment with 5-Aza-dC initiates demethylation by 12 h and transcription by 24 h at a hypermethylated hMLH1 promoter. RKO cells were treated with 1 μm 5-Aza-dC and harvested at the indicated time points. A, MSP analysis of hMLH1. A doubled line above the hMLH1 promoter schematic in Fig. 1A<$REFLINK> indicates the region examined by MSP. Methylation was detected by the presence of a PCR product amplified by methylation-specific primers in the M lanes. Demethylation was detected by PCR products amplified by unmethylated-specific primers in the U lanes. Bisulfite dH2O denotes bisulfite-treated dH2O, which served as a negative control for the treatment. RKO and SW480 served as positive controls for the methylated and unmethylated PCR reactions, respectively. B, reverse transcriptase-PCR analysis of hMLH1 expression. GAPDH expression served as a loading control. Five-day mock- and 5-day 5-Aza-dC-treated RKO cells served as negative and positive controls, respectively, for hMLH1 expression.
Tables
- Table 1
Summary of time course data
Changes observed 0 h 12 h 24 h 48 h 5 d DNA demethylation No Yes Yes Yes Yes Gene reexpression No No Yes Yes Yes Acetylated H3 ↓a ↓ ↓ ↑ ↑ Methyl-H3-K4 ↓ ↓ ↓ ↑ ↑ Methyl-H3-K9 ↑ ↑ ↑ ↓ ↓ -
a ↓, depletion; ↑, enrichment.
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Volume 62, Issue 24
