Methylation
Methylation, mutation, and cancer: new insights into the transcriptome
A review of Comparing the DNA Hypermethylome with Gene Mutations in Human Colorectal Cancer.
Note: This is a review of the published article listed below. All information, quotes, figures, methods, and findings mentioned in this review are from that article, and are the property of its authors and/or the publication in which the article originally appeared.
The loss of proper gene function during human cancer progression has been established to occur through both genetic and epigenetic mechanisms. Schuebel and colleagues (2007) developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. The researchers stratified genes according to altered signal intensity on Agilent’s 44K human microarrays to compare wild type (WT) and isogenic knockout counterparts for DNA methyltransferase 1 or 3b and identify global hypermethylation-dependent gene expression changes in human colorectal cancer cells (CRC). Agilent’s microarrays helped the group readily identify candidate cancer genes in single tumors with a high efficiency of validation, which can then be compared to mutated genes previously identified for CRC to establish key relationships between the altered tumor genome and hypermethylome. The research presented in this paper contributes to understanding the molecular pathways driving tumorigenesis; provides useful new DNA hypermethylation biomarkers to monitor cancer risk assessment, early diagnosis, and prognosis; and permits better monitoring of gene expression during cancer prevention and/or therapeutic strategies.
Figure 1. Approach for identification of the human cancer cell hypermethylome in HCT116 CRC cells.
(A) RNA from the indicated cell lines was isolated, labeled, hybridized, scanned, and fluorescent spot intensities normalized by background subtraction and Loess transformation using Agilent Technologies 44K human microarrays. Parental wild-type HCT116 cells (WT) and isogenic knockout counterparts for DNA methyltransferase 1 (DNMT1-/-) or 3b (DNMT3B-/-) are compared in our study. DKO cells are doubly deficient for both DNMT1 and DNMT3B. (B) Gene-expression changes in HCT116 cells with genetic disruption of various DNA methyltransferases. A 3-D scatter plot indicating the gene expression levels in HCT 116 cells with genetic disruption of DNMT1 (x-axis), DNMT3B (z-axis), and both DNMT1 and DNMT3B (DKO; y-axis) in fold scale. Individual gene-expression changes are in black with the average for three experiments (red spots) or from an individual experiment (blue spots) for those genes in DKO cells with greater than 4-fold expression change. (C) HCT116 cells were treated with 300 nM TSA for 18 h or 5 lM DAC for 96 h and processed as described above. (D) Gene-expression changes for HCT116 cells treated with TSA (x-axis) or DAC (y-axis) are plotted by fold change. Yellow spots indicate genes from DKO cells with 2-fold changes and above. Notice the loss of sensitivity when compared to gene-expression increases seen in DKO cells (80% of genes greater than 4-fold in the DKO cells now becomes greater than 1.3-fold in DAC-treated cells). Green spots indicate randomly selected genes verified to have complete promoter methylation in wild-type cells, reexpression in DKO cells and after DAC treatment, while red spots indicate selected genes that were identified as false positives (See Figures 4, 6, and 7 for validation results). Blue spots indicate the location of the 11 guide genes—previously shown to be hypermethylated and completely silenced in HCT 116 cells—used in this study (see Figure 3 for description). A distinct group of genes, including five of 11 guide genes, displays increases of greater than 2-fold after DAC treatment but no increase after TSA treatment. These genes form the top tier of candidate hypermethylated genes as discussed in the text. (E) Relatedness of whole-transcriptome expression patterns identified by dendrogram analysis. Individual single genetic disruption of DNMT1 and DNMT3B, DKO and DAC treatment, and TSA treatment each form three distinct categories of gene expression changes.
Figure 2. Characterization of the human cancer cell hypermethylome in different human CRC cell lines.
(A) Gene-expression changes for the indicated cells treated with TSA (x-axis) or DAC (y-axis) are plotted by log-fold change, and individual genes are shown in black. (B) Validation of the DNA hypermethylome. The characteristic spike of hypermethylated genes defined by treatment of cells with DAC or TSA consists of two tiers, with distinct features. The top tier of genes was identified as a zone in which gene expression did not increase with TSA (<1.4 fold) and displayed no detectable expression in wild-type cells, but increased greater than 2-fold with DAC treatment. The next tier of genes was identified as a cluster of genes for which expression changes of TSA and wild type were identical to those in the top tier, but increased between 1.4-fold and 2-fold with DAC treatment. Gene expression validation by RT-PCR and MSP indicated a validation frequency of 91% for top-tier genes in HCT116 cells, including genes that increased in DKO cells by greater than 2-fold. Next-tier genes in HCT116 cells were confirmed at a frequency of 49%, and in the SW480 top tier, with a frequency of 65%. (C) Shared candidate hypermethylated genes in CRC cell lines. We identified a total of 5,906 unique genes in all six cell lines with expression changes falling within the criteria of top- or next-tier categories. Overlaps in gene expression changes among two, three, four, five, or six cell lines are indicated; these range from 1,414 genes shared among two cell lines to 78 genes that were shared among all six cell lines.
Figure 3. Guide genes used in this study.
(A) Gene names, Agilent Technologies probe name, Genbank accession number, and references for the 11 guide genes previously shown to be hypermethylated and completely silenced in HCT116 cells. (B, C) Blue spots and gene names indicate the location of the 11 guide genes in a plot of TSA (x-axis) versus DAC (y-axis) gene expression changes on a log scale (B) or fold-change (C) scale. Five of 11 guide genes, circled in green, display increases of greater than 2-fold after DAC treatment but no increase after TSA treatment and these same genes have greater than 3-fold increases in DKO cells (green circle). (D) Direct comparison of guide genes in DKO and DAC plots. A distinct group of five guide genes, indicated by a green circle, showing greater than 3-fold expression changes in DKO cells and greater than 2-fold in DAC-treated cells, define the upper tier of candidate hypermethylated genes. Another three genes increased 1.3-fold, and three failed to increase with DAC treatment, allowing criteria for the next tier of gene expression.
Figure 4. Comparison of hypermethylation frequencies in human tumor samples.
Methylation analysis of verified hypermethylome genes in human tissue samples. Twenty genes from the verified gene lists were randomly selected from the HCT116 top tier (BOLL, DDX43, DKK3, FOXL2, HOXD1, JPH3, NEF3, NEURL, PPP1R14A, RAB32, STK31, and TLR2), HCT116 next tier (SALL4 and TP53AP1), or SW480 top tier (ZFP42) and analyzed for methylation in CRC cell lines (white columns), normal colon (red columns), or primary tumors (green columns). Percentage of methylation is indicated on the y-axis, and the abbreviated gene name on the x-axis. We tested at least six different cell lines, 16 to 40 colonic samples from noncancer patients, and between 18 and 61 primary CRC samples for each gene.
Title: Comparing the DNA Hypermethylome with Gene Mutations in Human Colorectal Cancer
Journal: PLoS Genet 3(9): e157.
Authors: Schuebel KE, Chen W, Cope L, Glöckner SC, Suzuki H, Yi JM, Chan TA, Neste LV, Criekinge WV, Bosch SV, van Engeland M, Ting AH, Jair K, Yu W, Toyota M, Imai K, Ahuja N, Herman JG, Baylin SB.
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