Genome-wide mapping and characterization of hypomethylated sites in human tissues and breast cancer cell lines - PubMed (original) (raw)

Genome-wide mapping and characterization of hypomethylated sites in human tissues and breast cancer cell lines

Yih-Jyh Shann et al. Genome Res. 2008 May.

Abstract

We have developed a method for mapping unmethylated sites in the human genome based on the resistance of TspRI-digested ends to ExoIII nuclease degradation. Digestion with TspRI and methylation-sensitive restriction endonuclease HpaII, followed by ExoIII and single-strand DNA nuclease allowed removal of DNA fragments containing unmethylated HpaII sites. We then used array comparative genomic hybridization (CGH) to map the sequences depleted by these procedures in human genomes derived from five human tissues, a primary breast tumor, and two breast tumor cell lines. Analysis of methylation patterns of the normal tissue genomes indicates that the hypomethylated sites are enriched in the 5' end of widely expressed genes, including promoter, first exon, and first intron. In contrast, genomes of the MCF-7 and MDA-MB-231 cell lines show extensive hypomethylation in the intragenic and intergenic regions whereas the primary tumor exhibits a pattern between those of the normal tissue and the cell lines. A striking characteristic of tumor cell lines is the presence of megabase-sized hypomethylated zones. These hypomethylated zones are associated with large genes, fragile sites, evolutionary breakpoints, chromosomal rearrangement breakpoints, tumor suppressor genes, and with regions containing tissue-specific gene clusters or with gene-poor regions containing novel tissue-specific genes. Correlation with microarray analysis shows that genes with a hypomethylated sequence 2 kb up- or downstream of the transcription start site are highly expressed, whereas genes with extensive intragenic and 3' untranslated region (UTR) hypomethylation are silenced. The method described herein can be used for large-scale screening of changes in the methylation pattern in the genome of interest.

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Figures

Figure 1.

Schematic diagram showing the strategy of mapping hypomethylated sequences in the genome by TspRI–HpaII–ExoIII digestion. Genomic DNA was first digested with TspRI, which created DNA fragments with a nine-base 3′ extension and were resistant for exonuclease III digestion. The DNA fragments were then digested with methylation-sensitive restriction enzyme HpaII. After treatment with exonuclease III and RecJf exonuclease, ExoIII-digested or mock-digested DNA was purified and labeled with Cy3 and Cy5 fluorescence dyes, respectively. Cy3 hybridization intensity was normalized to Cy5 for comparison among samples.

Figure 2.

Scatter plot of TspRI–ExoIII–array CGH data. (A) MCF7 genomic DNA digested with TspRI–HpaII–ExoIII; hybridization signals of the data points not containing HpaII sites flanked by TspRI sites are close to a ratio of one between ExoIII-digested and control mock-digested DNA, as expected since these TspRI fragments should not contain ExoIII-sensitive ends. The two lines correspond to ratios of four and 0.25, respectively, between the Cy3 and Cy5 signals. Some of the HpaII-sensitive background is probably due to sequence polymorphism creating new HpaII sites, as revealed by SssI and MspI experiments. (B) A large fraction of hybridization signal in the 53,728 TspRI fraction containing HpaII sites is lower after digestion with HpaII and ExoIII. (C) After SssI methylation of CpG sites in vitro, the 53,728 TspRI fragments containing HpaII sites became resistant to HpaII–ExoIII digestion with a hybridization ratio of around one in contrast to the result in B. (D,E) MCF7 genomic DNA digested with CpG-methylation–insensitive restriction enzyme MspI results in depletion of signals with a ratio of one in the fraction containing HpaII sites and a lowering of signals, whereas the fragments without HpaII sites are not affected significantly.

Figure 3.

Distribution of positions of hypomethylated sites in promoter, 1st exon, 1st intron, inner region of gene, and region containing no transcribed sequences: (A) MCF7, (B) MDA-MB-231, (C) tumor breast, (D) normal breast, and (E) normal brain. The extensively hypomethylated sites in MCF7 and MDA-MB-231 genomes are enriched in the intragenic and intergenic regions (P < 0.01, Fisher’s exact test). By contrast, normal tissue genomes are enriched in the promoter, exon 1, and intron 1 close to the 5′ end of the gene (P < 0.01, Fisher’s exact test).

Figure 4.

Megabase-sized hypomethylated zones in tumor cell genomes associated with fragile sites. Red and green lines represent the hypomethylated zones in MCF7 and MDA-MB-231, respectively. Blue lines represent 86 common fragile sites in human genome. We found that 79% of fragile sites were in the hypomethylated zones in the MCF-7 genome (P < 0.01), and this correlation was also significant in the MDA-MB-231 genome (P < 0.01).

Figure 5.

Megabase-sized hypomethylation zones in tumor cells. The majority of large hypomethylated zones in the MCF7 genome occur in the genomic region with low gene density. (A) Hypomethylated zones in breast tumor genomes are located in low-gene-density regions in chromosome 1 (lowest line). In MCF-7 and MDA-MB-231 genomes, the hypomethylated zones are in association with these low-gene-density regions, and the _P_-values of Fisher’s exact test are <0.01. Arrows represent the hypomethylated fragile sites in MCF-7 cell line genome, FRA1A (1p35–36.1), FRA1B (DAB1), FRA1C (1p31.2), FRA1E (DPYD), FRA1G (1q25.1), and FRA1H (USH2A and ESRRG). (B) The 12q24.31–32 hypomethylated regions (white bars) are found in the genomes of MCF-7 (P < 0.01), MDA-MB-231 (P < 0.01), and primary breast tumor (P < 0.01) as compared with normal breast genome. The red horizontal lines represent ratios of one between signals of ExoIII-digested and mock-digested DNA.

Figure 6.

Megabase-sized hypomethylation zones which contain clusters of tissue-specific genes in tumor cells. As compared with normal breast genome, the 1q23 hypomethylated region is found in the genomes of MCF-7 and primary breast tumor (P < 0.01) and less prominently in MDA-MB-231 genome (P = 0.60). The red horizontal lines represent ratios of one between signals of ExoIII-digested and mock-digested DNA.

Figure 7.

Box plot of correlation between position of DNA hypomethylation and gene expression. Gene expression level obtained from Affymetrix analysis is correlated with hypomethylated sites at every kb up- or downstream from transcription initiation site. (A) Hypomethylation 2 kb up- or downstream from transcription start site is correlated with higher gene expression (**P < 0.01, _t_-test). The red line represents the median expression level for each group of genes with hypomethylated sites located 1–5 kb up- or downstream from the transcription start site. (B) Stand-alone promoter has a high level of expression. (C,D) Genes with extensive intragenic hypomethylation and hypomethylation at 3′ UTR show low expression (**P < 0.01, _t_-test).

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Genome-wide mapping and characterization of hypomethylated sites in human tissues and breast cancer cell lines - PubMed (original) (raw)