Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation-based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAF V600E mutation. A CIMPlow (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent~7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing.

Aberrant DNA methylation patterns, including global hypomethylation, gene-specific hypermethylation/hypomethylation, and loss of imprinting (LOI), are common in acute myelogenous leukemia (AML) and other cancer tissues. We investigated for the first time whether such epigenetic changes are induced in healthy subjects by low-level exposure to benzene, a widespread pollutant associated with AML risk. Blood DNA samples and exposure data were obtained from subjects with different levels of benzene exposure, including 78 gas station attendants, 77 traffic police officers, and 58 unexposed referents in Milan, Italy (personal airborne benzene range, <6-478 Mg/m 3 ). Bisulfite-PCR pyrosequencing was used to quantitate DNA methylation in long interspersed nuclear element-1 (LINE-1) and AluI repetitive elements as a surrogate of genome-wide methylation and examine genespecific methylation of MAGE-1 and p15. Allele-specific pyrosequencing of the H19 gene was used to detect LOI in 96 subjects heterozygous for the H19 imprinting center G/A single-nucleotide polymorphism. Airborne benzene was associated with a significant reduction in LINE-1 (À2.33% for a 10-fold increase in airborne benzene levels; P = 0.009) and AluI (À1.00%; P = 0.027) methylation. Hypermethylation in p15 (+0.35%; P = 0.018) and hypomethylation in MAGE-1 (À0.49%; P = 0.049) were associated with increasing airborne benzene levels. LOI was found only in exposed subjects (4 of 73, 5.5%) and not in referents (0 of 23, 0.0%). However, LOI was not significantly associated with airborne benzene (P > 0.20). This is the first human study to link altered DNA methylation, reproducing the aberrant epigenetic patterns found in malignant cells, to low-level carcinogen exposure. [Cancer Res 2007;67(3):876-80]

Rationale: Prenatal exposure to tobacco smoke increases the risk for diseases later in the child's life that may be mediated through alterations in DNA methylation. Objectives: To demonstrate that differences in DNA methylation patterns occur in children exposed to tobacco smoke and that variation in detoxification genes may alter these associations. Methods: Methylation of DNA repetitive elements, LINE1 and AluYb8, was measured using bisulfite conversion and pyrosequencing in buccal cells of 348 children participating in the Children's Health Study. Gene-specific CpG methylation differences associated with smoke exposure were screened in 272 participants in the Children's Health Study children using an Illumina GoldenGate panel. CpG loci that demonstrated a statistically significant difference in methylation were validated by pyrosequencing. Estimates were standardized across loci using a Z score to enable crosscomparison of results. Measurements and Main Results: DNA methylation patterns were associated with in utero exposure to maternal smoking. Exposed children had significantly lower methylation of AluYb8 (b, 20.31; P 5 0.03). Differences in smoking-related effects on LINE1 methylation were observed in children with the common GSTM1 null genotype. Differential methylation of CpG loci in eight genes was identified through the screen. Two genes, AXL and PTPRO, were validated by pyrosequencing and showed significant increases in methylation of 0.37 (P 5 0.005) and 0.34 (P 5 0.02) in exposed children. The associations with maternal smoking varied by a common GSTP1 haplotype. Conclusions: Life-long effects of in utero exposures may be mediated through alterations in DNA methylation. Variants in detoxification genes may modulate the effects of in utero exposure through epigenetic mechanisms.

DNA methylation is known to be associated with cell differentiation, aging, disease and cancer. There exists an expanding base of knowledge regarding tissue-specific DNA methylation, but we have little information about person-specific DNA methylation. Here, we analyze the DNA methylation patterns of multiple tissues from multiple individuals using a high-throughput quantitative assay of genome-wide DNA methylation, namely the Illumina GoldenGate BeadArray. DNA methylation patterns were largely conserved across 11 different tissues (r = 0.852) and across six individuals (r = 0.829), and we found that DNA was highly methylated in non-CpG islands and/or CpG sites that are not occupied by either H3K4me3 or H3K27me3 (P < 0.05). Finally, we found that the Illumina GoldenGate assay features a large number of probes (265/1505 probes, 17.6%) that contain single-nucleotide polymorphisms, which may interfere with DNA methylation analyses in genome-wide studies.

It was recently shown that a large portion of the human transcriptome can originate from within repetitive elements, leading to ectopic expression of protein-coding genes. However the mechanism of transcriptional activation of repetitive elements has not been definitively elucidated. For the first time, we directly demonstrate that hypomethylation of retrotransposons can cause altered gene expression in humans. We also reveal that active LINE-1s switch from a tetranucleosome to dinucleosome structure, acquiring H2A.Z- and nucleosome-free regions upstream of TSSs, previously shown only at active single-copy genes. Hypomethylation of a specific LINE-1 promoter was also found to induce an alternate transcript of the MET oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. These data show that, in addition to contributing to chromosomal instability, hypomethylation of LINE-1s can alter the functional transcriptome and plays a role not only in human disease but also in disease predisposition.