Genome-wide alterations in gene methylation by the BRAF V600E mutation in papillary thyroid cancer cells

The BRAF V600E mutation plays an important role in the tumorigenesis of papillary thyroid cancer (PTC). To explore an epigenetic mechanism involved in this process, we performed a genome-wide DNA methylation analysis using a methylated CpG island amplification (MCA)/CpG island microarray system to examine gene methylation alterations after shRNA knockdown of BRAF V600E in thyroid cancer cells. Our results revealed numerous methylation targets of BRAF V600E mutation with a large cohort of hyper- or hypo-methylated genes in thyroid cancer cells, which are known to have important metabolic and cellular functions. As hypomethylation of numerous genes by BRAF V600E was particularly a striking finding, we took a further step to examine the selected 59 genes that became hypermethylated in both cell lines upon BRAF V600E knockdown and found them to be mostly correspondingly under-expressed (i.e. they were normally maintained hypomethylated and over-expressed by BRAF V600E in thyroid cancer cells). We confirmed the methylation status of selected genes revealed on MCA/CpG microarray analysis by performing methylation-specific PCR. To provide proof of concept that some of the genes uncovered here may play a direct oncogenic role, we selected six of them to perform shRNA knockdown and examined its effect on cellular functions. Our results demonstrated that the HMGB2 gene played a role in PTC cell proliferation and the FDG1 gene in cell invasion. Thus, this study uncovered a prominent epigenetic mechanism through which BRAF V600E can promote PTC tumorigenesis by altering the methylation and hence the expression of numerous important genes.     

Microarray-based method for detecting methylation changes of p16^Ink4a gene 5＇-CpG islands in gastric carcinomas

AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high-throughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer. METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP). RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover, it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP. CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.     

Epigenetic-aging-signature to determine age in different tissues

All tissues of the organism are affected by aging. This process is associated with epigenetic modifications such as methylation changes at specific cytosine residues in the DNA (CpG sites). Here, we have identified an Epigenetic-Aging-Signature which is applicable for many tissues to predict donor age. DNA-methylation profiles of various cell types were retrieved from public data depositories - all using the HumanMethylation27 BeadChip platform which represents 27,578 CpG sites. Five datasets from dermis, epidermis, cervical smear, T-cells and monocytes were used for Pavlidis Template Matching to identify 19 CpG sites that are continuously hypermethylated upon aging (R>0.6; p-value<10-13). Four of these CpG sites (associated with the genes NPTX2, TRIM58, GRIA2 and KCNQ1DN) and an additional hypomethylated CpG site (BIRC4BP) were implemented in a model to predict donor age. This Epigenetic-Aging-Signature was tested on a validation group of eight independent datasets corresponding to several cell types from different tissues. Overall, the five CpG sites revealed age-associated DNA-methylation changes in all tissues. The average absolute difference between predicted and real chronological age was about 11 years. This method can be used to predict donor age in various cell preparations - for example in forensic analysis.     

Genome-wide DNA methylation profiles in hepatocellular carcinoma

Alterations in DNA methylation frequently occur in hepatocellular cancer (HCC). We have previously demonstrated that hypermethylation in candidate genes can be detected in plasma DNA before HCC diagnosis. To identify, with a genome-wide approach, additional genes hypermethylated in HCC that could be used for more accurate analysis of plasma DNA for early diagnosis, we analyzed tumor and adjacent nontumor tissues from 62 Taiwanese HCC cases using Illumina methylation arrays (Illumina, Inc., San Diego, CA) that screen 26,486 autosomal CpG sites. After Bonferroni adjustment, a total of 2,324 CpG sites significantly differed in methylation level, with 684 CpG sites significantly hypermethylated and 1,640 hypomethylated in tumor, compared to nontumor tissues. Array data were validated with pyrosequencing in a subset of five of these genes; correlation coefficients ranged from 0.92 to 0.97. Analysis of plasma DNA from 38 cases demonstrated that 37%-63% of cases had detectable hypermethylated DNA (≥ 5% methylation) for these five genes individually. At least one of these genes was hypermethylated in 87% of the cases, suggesting that measurement of DNA methylation in plasma samples is feasible. CONCLUSION: The panel of methylated genes indentified in the current study will be further tested in a large cohort of prospectively collected samples to determine their utility as early biomarkers of HCC.     

High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer

Changes in DNA methylation patterns are an important characteristic of human cancer. Tumors have reduced levels of genomic DNA methylation and contain hypermethylated CpG islands, but the full extent and sequence context of DNA hypomethylation and hypermethylation is unknown. Here, we used methylated CpG island recovery assay-assisted high-resolution genomic tiling and CpG island arrays to analyze methylation patterns in lung squamous cell carcinomas and matched normal lung tissue. Normal tissues from different individuals showed overall very similar DNA methylation patterns. Each tumor contained several hundred hypermethylated CpG islands. We identified and confirmed 11 CpG islands that were methylated in 80-100% of the SCC tumors, and many hold promise as effective biomarkers for early detection of lung cancer. In addition, we find that extensive DNA hypomethylation in tumors occurs specifically at repetitive sequences, including short and long interspersed nuclear elements and LTR elements, segmental duplications, and subtelomeric regions, but single-copy sequences rarely become demethylated. The results are consistent with a specific defect in methylation of repetitive DNA sequences in human cancer.     

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.     

Hypermethylation of E-cadherin in leukemia

E-cadherin gene is often termed a "metastasis suppressor" gene because the E-cadherin protein can suppress tumor cell invasion and metastasis. Inactivation of the E-cadherin gene occurs in undifferentiated solid tumors by both genetic and epigenetic mechanisms; however, the role of E-cadherin in hematologic malignancies is only now being recognized. E-cadherin expression is essential for erythroblast and normoblast maturation, yet expression is reduced or absent in leukemic blast cells. This study examined the messenger RNA (mRNA) and protein expression of the E-cadherin gene in bone marrow and blood samples from normal donors and patients with leukemia. We found that all normal donor samples expressed E-cadherin mRNA, whereas both samples of acute myelogenous leukemia and chronic lymphocytic leukemia had a significant reduction or absence of expression. However, normal blast counterparts expressed only a low level of E-cadherin surface protein. Sodium bisulphite genomic sequencing was used to fully characterize the methylation patterns of the CpG island associated with the E-cadherin gene promoter in those samples with matched DNA. All of the normal control samples were essentially unmethylated; however, 14 of 18 (78%) of the leukemia samples had abnormal hypermethylation of the E-cadherin CpG island. In fact both alleles of the E-cadherin gene were often hypermethylated. We conclude the E-cadherin gene is a common target for hypermethylation in hematologic malignancies.     

Identification of the genomic region under epigenetic regulation during non‐alcoholic fatty liver disease progression

Aim

The progression of non‐alcoholic fatty liver disease (NAFLD) is affected by epigenetics. We undertook co‐methylation and differentially methylated region (DMR) analyses to identify the genomic region that is under epigenetic regulation during NAFLD progression.

Methods

We collected liver biopsy specimens from 60 Japanese patients with NAFLD and classified these into mild (fibrosis stages 0–2) or advanced (fibrosis stages 3–4) NAFLD. We carried out a genome‐wide DNA methylation analysis and identified the differentially methylated CpGs between mild and advanced NAFLD. Differentially methylated regions with multiple consecutive differentially methylated CpGs between mild and advanced NAFLD were extracted.

Results

Co‐methylation analysis showed that individual differentially methylated CpG sites were clustered into three modules. The CpG sites clustered in one module were hypomethylated in advanced NAFLD and their annotated genes were enriched for “immune system” function. The CpG sites in another module were hypermethylated and their annotated genes were enriched for “mitochondria” or “lipid particle”, and “lipid metabolism” or “oxidoreductase activity”. Hypomethylated DMRs included tumorigenesis‐related genes (FGFR2, PTGFRN, and ZBTB38), the expressions of which are upregulated in advanced NAFLD. Tumor suppressor MGMT had two DMRs and was downregulated. Conversely, FBLIM1 and CYR61, encoding proteins that reduce cell proliferation, showed hypomethylated DMRs and were upregulated. Expression of the antioxidant gene NQO1 was upregulated, with a hypomethylated DMR. The DMR containing cancer‐related MIR21 was hypomethylated in advanced NAFLD.

Conclusions

Co‐methylation and DMR analyses suggest that the NAFLD liver undergoes mitochondrial dysfunction, decreased lipid metabolism, and impaired oxidoreductase activity, and acquires tumorigenic potential at the epigenetic level.     

Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells

OBJECTIVE: To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA). METHODS: Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques. RESULTS: The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression. Furthermore, the expression of DR-3 protein was down-modulated in association with methylation of the promoter CpG island in RA synovial cells. CONCLUSION: The CpG island in the DR3 gene promoter was specifically methylated to down-modulate the expression of DR-3 protein in rheumatoid synovial cells, which may provide resistance to apoptosis in RA synovial cells.