Genes showing a negative correlation between gene expression and DNA methylation.

<p>Average β-values are plotted on the x-axis, log2 gene expression values on the y-axis. Dots represent a cell line; <i>BRAF</i> mutant cell lines are marked with an x.</p

Methylation associated transcriptional repression of <i>ELOVL5</i> in novel colorectal cancer cell lines

<div><p>Genetic and epigenetic alterations mark colorectal cancer (CRC). Global hypomethylation is observed in nearly all CRC, but a distinct subset of CRC show the CpG Island Methylator Phenotype (CIMP). These tumors show DNA hypermethylation of a specific subset of CpG islands, resulting in transcriptional downregulation of nearby genes. Recently we reported the establishment of novel CRC cell lines derived from primary and metastatic CRC tissues. In this study we describe the DNA methylation profiling of these low passage CRC cell lines. We generated global DNA methylation profiles with Infinium HumanMethylation450 BeadChips and analysed them in conjunction with matching gene expression profiles. Multidimensional scaling of the DNA methylation and gene expression datasets showed that <i>BRAF</i> mutated cell lines form a distinct group. In this group we investigated the 706 loci which we have previously identified to be hypermethylated in <i>BRAF</i> mutant CRC. We validated the significant findings in the The Cancer Genome Atlas colon adenocarcinoma dataset. Our analysis identified <i>ELOVL5</i>, <i>FAM127B</i>, <i>MTERF1</i>, <i>ZNF606</i> to be subject to transcriptional downregulation through DNA hypermethylation in CRC. We further investigated <i>ELOVL5</i> with qPCR and immunohistochemical staining, validating our results, but did not find a clear relation between ELOVL5 expression and tumor stage or relapse free survival. <i>ELOVL5</i>, <i>FAM127B</i>, <i>MTERF1</i>, <i>ZNF606</i> are involved in important cellular processes such as apoptosis, lipogenesis and the downstream transcriptional effect of the MAPK-pathway. We have identified a DNA methylation profile regulating key cellular processes in CRC, resulting in a growth advantage to the tumor cells.</p></div

Genes showing correlation between expression and methylation in the TCGA data.

<p>RSEM gene expression values were plotted against the average β-values of the methylation loci.</p

ELOVL5 immunohistochemical staining results.

<p>ELOVL5 immunohistochemical staining results.</p

Clustering of gene expression and DNA methylation data.

<p>To minimize overlap between cell line names, only the numbers corresponding to the cell lines were displayed. MDS analysis of DNA methylation data shows a distinct cluster of <i>BRAF</i> mutant cell lines; cluster C. Secondary clustering is formed by <i>KRAS</i> mutant samples with low <i>ZNF304</i> expression (cluster B, Figure 2A). MDS analysis of gene expression data showed <i>BRAF</i> mutant samples cluster separately from <i>KRAS</i> mutant and WT samples (2B). The cell lines in cluster B show a 5.9-fold lower <i>ZNF304</i> expression compared to the cell lines in cluster A (2C).</p

Regulation and targeting of CIMP profiles by MAFG and ZNF304.

<p>Mutant KRAS upregulates of deubiquitinase USP28, leading to ZNF304 upregulation. ZNF304 recruits a corepressor complex which subsequently methylates the nearby CpGI. 1B: MAFG is upregulated and phosphorylated through MAPK-signalling which when bound to it’s binding site recruits a corepressor complex resulting in DNA hypermethylation (1B). Overview of DNA methylation outcomes for genes with and without MAFG and ZNF304 binding sites in CIMP0, CIMP1 and CIMP2 tumors (1C). bs = binding site, TSS = Transcription Start Site, CpGI = CpG Island.</p

<i>CD1D</i> expression analyses in patient samples.

<p>qRT-PCR expression analysis of <i>CD1D</i> in 184 paired carcinoma and normal samples. A strong transcriptional downregulation was observed in the carcinoma compared to the normal samples. Gene expression values are relative to the median of the normal samples (4A). Comparison of <i>CD1D</i> expression in 34 polyp samples compared to the 184 normal and carcinoma samples. <i>CD1D</i> is also downregulated in the polyps, but downregulation in polyps is not as strong as in the carcinoma. Gene expression values are relative to the median of the normal samples. The box represent the 25-75-quartiles, the whiskers represent the 5–95% percentiles (4B). Comparison of the <i>CD1D</i> expression normal/carcinoma ratio in methylated versus unmethylated carcinoma. <i>CD1D</i> hypermethylation does not affect the degree of transcriptional downregulation. The bars represent the mean with 95% confidence interval (4C).</p

Gene expression of <i>MEIS1</i> and <i>MEIS1D</i>_<i>27</i> expression in human colorectal samples and CRC cell lines.

<p><i>MEIS1</i> gene expression as measured by RT-qPCR corrected for the geometric mean of the housekeeping genes <i>CPSF6</i> and <i>HNRNPM</i>. (<b>A</b>) <i>BRAF</i>^p.V600E tumors (black bars) that were all <i>MEIS1</i> methylated showed lower expression of the full length <i>MEIS1</i> gene, when compared to the paired normal tissues (white bars). (<b>B</b>) Colorectal cancer cell lines that were methylated for the <i>MEIS1</i> gene promoter were devoid of <i>MEIS1</i> gene expression. HT29, LS411N and RKO colon cancer cell lines carried the <i>BRAF</i>^p.V600E mutation, whereas the remaining cell lines were wild type for <i>BRAF</i>. (<b>C</b>) Gene expression of the truncated <i>MEIS1D</i>_<i>27</i> transcript in <i>BRAF</i>^p.V600E tumors (black bars) was low to absent when compared to paired normal tissue (white bars). Primer sets used, uniquely detect the truncated transcript. (<b>D</b>) <i>MEIS1</i> methylated colon cancer cells were devoid of <i>MEIS</i>_<i>D27</i> (i.e. exon 8 skipped <i>MEIS1</i>). Gene expression was expressed relative to SW837.</p

<i>MEIS1</i> is methylated in <i>BRAF</i>^p.V600E relative to <i>BRAF</i> wild type tumors.

<p>(<b>A</b>) The <i>MEIS1</i> promoter is hypermethylated in colorectal tumors with a <i>BRAF</i>^p.V600E mutation (black dots) when compared to wild type <i>BRAF</i> (white dots). The Y-axis represents the tumor vs. normal log₂ ratio for the median probe per CpG fragment. The horizontal dotted line at log₂ ratio 0 indicates an equal extent of <i>MEIS1</i> methylation in tumor and normal samples. (<b>B</b>) Overview of the analyzed <i>MEIS1</i> promoter, CpG islands within the promoter and the locus analyzed by MSP primers. Locations were based on the human genome browser (UCSC assembly March 2006, hg18). (<b>C</b>) <i>MEIS1</i>-MSP data showing hypermethylation in <i>BRAF</i>^p.V600E colorectal tumors when compared to <i>BRAF</i> wild types. T: tumor; N: normal tissue; M: methylated <i>MEIS1</i> promoter (168 bp); Um: Unmethylated <i>MEIS1</i> promoter (176 bp).</p

Additional file 1 of Enrichment of colibactin-associated mutational signatures in unexplained colorectal polyposis patients

Supplementary Material 1: Somatic variants detected using targeted NGS fitting SBS88 in other genes than AP