Department of Biochemistry, College of Life Science and Technology, Yonsei University, Seoul, Korea.
Abstract
BACKGROUND Epigenetic alteration of gene expression is a common event in human cancer. DNA methylation is a well-known epigenetic process, but verifying the exact nature of epigenetic changes associated with cancer remains difficult.
METHODS We profiled the methylome of human gastric cancer tissue at 50-bp resolution using a methylated DNA enrichment technique (methylated CpG island recovery assay) in combination with a genome analyzer and a new normalization algorithm.
RESULTS We were able to gain a comprehensive view of promoters with various CpG densities, including CpG Islands (CGIs), transcript bodies, and various repeat classes. We found that gastric cancer was associated with hypermethylation of 5' CGIs and the 5'-end of coding exons as well as hypomethylation of repeat elements, such as short interspersed nuclear elements and the composite element SVA. Hypermethylation of 5' CGIs was significantly correlated with downregulation of associated genes, such as those in the HOX and histone gene families. We also discovered long-range epigenetic silencing (LRES) regions in gastric cancer tissue and identified several hypermethylated genes (MDM2, DYRK2, and LYZ) within these regions. The methylation status of CGIs and gene annotation elements in metastatic lymph nodes was intermediate between normal and cancerous tissue, indicating that methylation of specific genes is gradually increased in cancerous tissue.
CONCLUSIONS Our findings will provide valuable data for future analysis of CpG methylation patterns, useful markers for the diagnosis of stomach cancer, as well as a new analysis method for clinical epigenomics investigations.
Figures
Figure 1Methylation patterns of normal gastric tissue. (A) Chromosome-wide average MES is shown as a function of the average CpG density, gene density (the number of genes per Mb), LINE quantity (the length of LINE per Mb), and SINE quantity (the length of SINE per Mb) for each chromosome. (B) For chromosome 22, the average CpG density (shaded gray) and MES (black curve) were obtained in 1-Mb sliding windows. The positions of transcribed genes (black bars at the bottom), CG islands (blue bars at the top), and long repeats (> 1 kb; red bars on the top) are compared against the backdrop of DNA methylation and CpG density (left). The average MES for CGIs, gene bodies, and repeats (right). (C) The distribution of gene bodies and CGI MES (left). The average MES for promoter-associated and promoter-independent CGIs is shown to the right. (D) The average MES for promoter subgroups, based on the existence of CGI (left). (E) Basic information on intergenic, exonic, and intronic regions, according to length, CpG number, and mapped reads (left). The distribution of intergenic, exonic, and intronic MESs is shown to the right. (F) Basic information on the upstream 1-kb region, 5' UTR exons, coding exons, 3' UTR exons, and downstream 1-kb region according to length, CpG number, and mapped reads (left). The distribution of the MES for each element is shown to the right.Figure 2Comparison of methylation patterns in normal and cancerous tissue. (A) Average MES curve for normal (black) and cancerous (red) tissue in chromosome 19 (left). The average MES for CGIs, gene bodies, and repeats (right).(B) DNA methylation of gene annotated elements. Each element (upstream 1 kb, exon, intron, and downstream 1 kb) were partitioned into 20 bins and the average MES was obtained for each bin of all corresponding elements. (C) DNA methylation at transcript ends and coding region ends. The average MES was obtained in a sliding 50-bp window according to its distance from the transcript start (first) and end (second) for CGI-positive promoters as well as the transcript start (third) and end (fourth) for CGI-positive promoters. (D) DNA methylation of total 5' UTR exons (left) and 5' UTR coding exons (right).Figure 3DNA methylation of CpG islands. (A) Relative differential MES of subgrouped CGIs. (B) Correlation between differential CGI methylation and distance to the transcription start site. (C) Correlation between gene expression level and the hypermethylation of CGIs. (D) Methylation-specific PCR of histone genes showing the highest differential MES values. M1 and U1 correspond to HIST3H2A, while M2 and U2 correspond to HIST3H2B.Figure 4DNA methylation of repetitive elements. (A) Differential MES (expressed as Cancer MES - Normal MES) for repeats of various classes. (B) Differential MES for Alu subfamilies. (C) Differential MES for repetitive elements grouped according to their position in the genome.Figure 5DNA methylation of LRES regions. (A) Average MES curve for LRES regions in chromosome 12 in normal (black) and cancerous (red) tissue. (B) Average MESs for gene promoters in LRES regions in normal and cancerous tissue. (C) Correlation between gene expression levels and hypermethylation of genes within LRES regions. (D) Methylation enrichment of several selected genes within LRES regions, as assessed by MIRA-qPCR. (E) Methylation ratio at the target site within the upstream region of MDM2, as measured by pyrosequencing.Figure 6Comparison of methylation patterns in normal and metastatic lymph nodes. DNA methylation at transcript ends and coding region ends is shown.
