Vitamin C promotes widespread yet specific DNA demethylation of the epigenome in human embryonic stem cells.

Vitamin C (ascorbate) is a widely used medium supplement in embryonic stem cell culture. Here, we show that ascorbate causes widespread, consistent, and remarkably specific DNA demethylation of 1,847 genes in human embryonic stem cells (hESCs), including important stem cell genes, with a clear bias toward demethylation at CpG island boundaries. We show that a subset of these DNA demethylated genes displays concomitant gene expression changes and that the position of the demethylated CpGs relative to the transcription start site is correlated to such changes. We further show that the ascorbate-demethylated gene set not only overlaps with gene sets that have bivalent marks, but also with the gene sets that are demethylated during differentiation of hESCs and during reprogramming of fibroblasts to induced pluritotent stem cells (iPSCs). Our data thus identify a novel link between ascorbate-mediated signaling and specific epigenetic changes in hESCs that might impact on pluripotency and reprogramming pathways.

Functional analysis of 5-lipoxygenase promoter repeat variants.

Variants of a hexanucleotide repeat polymorphism in the promoter of the 5-lipoxygenase (5-LO) gene have been associated with cardiovascular disease traits in humans, which may be due, at least in part, to differential expression of the at-risk alleles. To more fully characterize these variants, we carried out gene expression and DNA methylation studies in primary leukocytes from healthy individuals carrying various 5-LO promoter alleles. Regardless of genotype, 5-LO and 5-LO-activating protein (FLAP) gene expression was higher in granulocytes compared with monocytes and lymphocytes, whereas leukotriene A4 hydrolase (LTA4H) expression was higher in monocytes. In all three leukocyte populations, 5-LO mRNA levels were positively correlated with those of FLAP and LTA4H, with the highest correlation observed in granulocytes. In lymphocytes, individuals homozygous for the shorter 3 and 4 repeat alleles had between 20-35% higher 5-LO, FLAP and LTA4H expression compared with homozygous carriers of the wild-type 5 repeat allele (P = 0.03-0.0001). DNA methylation analysis of four CpG islands in a 1500 bp region encompassing the 5-LO promoter and the first approximately 100 bp of intron 1 revealed relatively low overall DNA methylation across all genotypes and leukocyte populations. However, analysis of the promoter repeats themselves demonstrated that, regardless of cell population, the 4 allele was methylated approximately twice as much as the 3 allele (P < 0.0001). Our results demonstrate that, in lymphocytes, the shorter repeat alleles of the 5-LO promoter lead to higher gene expression, which may be regulated through differential DNA methylation of the CpGs located within these repeats.

Epigenetic modification of CCAAT/enhancer binding protein alpha expression in acute myeloid leukemia.

Functional loss of CCAAT/enhancer binding protein alpha (C/EBP alpha), a master regulatory transcription factor in the hematopoietic system, can result in a differentiation block in granulopoiesis and thus contribute to leukemic transformation. Here, we show the effect of epigenetic aberrations in regulating C/EBP alpha expression in acute myeloid leukemia (AML). Comprehensive DNA methylation analyses of the CpG island of C/EBP alpha identified a densely methylated upstream promoter region in 51% of AML patients. Aberrant DNA methylation was strongly associated with two generally prognostically favorable cytogenetic subgroups: inv(16) and t(15;17). Surprisingly, while epigenetic treatment increased C/EBP alpha mRNA levels in vitro, C/EBP alpha protein levels decreased. Using a computational microRNA (miRNA) prediction approach and functional studies, we show that C/EBP alpha mRNA is a target for miRNA-124a. This miRNA is frequently silenced by epigenetic mechanisms in leukemia cell lines, becomes up-regulated after epigenetic treatment, and targets the C/EBP alpha 3' untranslated region. In this way, C/EBP alpha protein expression is reduced in a posttranscriptional manner. Our results indicate that epigenetic alterations of C/EBP alpha are a frequent event in AML and that epigenetic treatment can result in down-regulation of a key hematopoietic transcription factor.

Genome-epigenome interactions in cancer.

Genetic and epigenetic mechanisms contribute to the development of human tumors. However, the conventional analysis of neoplasias has preferentially focused on only one of these processes. This approach has led to a biased, primarily genetic view, of human tumorigenesis. Epigenetic alterations, such as aberrant DNA methylation, are sufficient to induce tumor formation, and can modify the incidence, and determine the type of tumor which will arise in genetic models of cancer. These observations raise important questions about the degree to which genetic and epigenetic mechanisms cooperate in human tumorigenesis, the identity of the specific cooperating genes and how these genes interact functionally to determine the diverse biological and clinical paths to tumor initiation and progression. These gaps in our knowledge are, in part, due to the lack of methods for full-scale integrated genetic and epigenetic analyses. The ultimate goal to fill these gaps would include sequencing relevant regions of the 3-billion nucleotide genome, and determining the methylation status of the 28-million CpG dinucleotide methylome at single nucleotide resolution in different types of neoplasias. Here, we review the emergence and advancement of technologies to map ever larger proportions of the cancer methylome, and the unique discovery potential of integrating these with cancer genomic data. We discuss the knowledge gained from these large-scale analyses in the context of gene discovery, therapeutic application and building a more widely applicable mechanism-based model of human tumorigenesis.

Aberrant DNA methylation of OLIG1, a novel prognostic factor in non-small cell lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. Currently, tumor, node, metastasis (TNM) staging provides the most accurate prognostic parameter for patients with non-small cell lung cancer (NSCLC). However, the overall survival of patients with resectable tumors varies significantly, indicating the need for additional prognostic factors to better predict the outcome of the disease, particularly within a given TNM subset. METHODS AND FINDINGS: In this study, we investigated whether adenocarcinomas and squamous cell carcinomas could be differentiated based on their global aberrant DNA methylation patterns. We performed restriction landmark genomic scanning on 40 patient samples and identified 47 DNA methylation targets that together could distinguish the two lung cancer subgroups. The protein expression of one of those targets, oligodendrocyte transcription factor 1 (OLIG1), significantly correlated with survival in NSCLC patients, as shown by univariate and multivariate analyses. Furthermore, the hazard ratio for patients negative for OLIG1 protein was significantly higher than the one for those patients expressing the protein, even at low levels. CONCLUSIONS: Multivariate analyses of our data confirmed that OLIG1 protein expression significantly correlates with overall survival in NSCLC patients, with a relative risk of 0.84 (95% confidence interval 0.77-0.91, p < 0.001) along with T and N stages, as indicated by a Cox proportional hazard model. Taken together, our results suggests that OLIG1 protein expression could be utilized as a novel prognostic factor, which could aid in deciding which NSCLC patients might benefit from more aggressive therapy. This is potentially of great significance, as the addition of postoperative adjuvant chemotherapy in T2N0 NSCLC patients is still controversial.

Epigenetic regulation of the tumor suppressor gene TCF21 on 6q23-q24 in lung and head and neck cancer.

The identification of tumor suppressor genes has classically depended on their localization within recurrent regions of loss of heterozygosity. According to Knudson's two-hit hypothesis, the remaining allele is lost, either genetically or, more recently identified, through epigenetic events. To date, retrospective analyses have determined promoter methylation as a common alternative alteration in cancer cells to silence cancer-related genes. Here we report an application of restriction landmark genomic scanning that allows for DNA methylation profiling along a region of recurrent loss of heterozygosity at chromosome 6q23-q24. This approach resulted in the identification of a tumor suppressor gene, TCF21, which is frequently lost in human malignancies. We demonstrate that TCF21 is expressed in normal lung airway epithelial cells and aberrantly methylated and silenced in the majority of head and neck squamous cell carcinomas and non-small-cell lung cancers analyzed. TCF21 is known to regulate mesenchymal cell transition into epithelial cells, a property that has been shown to be deficient in carcinomas. We further demonstrate that exogenous expression of TCF21 in cells that have silenced the endogenous TCF21 locus resulted in a reduction of tumor properties in vitro and in vivo.

Quantification of DNA methylation in electrofluidics chips (Bio-COBRA).

Alterations of normal gene expression patterns are a hallmark of human cancers. It is now clear that the dysregulation of epigenetic modifications of the DNA and surrounding histones contributes to aberrant gene silencing, thus being major participants not only in the progression but also the initiation of the disease phenotype. The best-studied epigenetic modification is DNA methylation, which converts cytosine to 5-methylcytosine. Aberrant hypermethylation of the promoter is frequently observed in cancer and is generally associated with gene silencing. Currently, accurate and reproducible quantification of DNA methylation remains challenging. Here, we describe Bio-COBRA, a modified protocol for Combined Bisulfite Restriction Analysis (COBRA), that incorporates an electrophoresis step in microfluidics chips. Microfluidics technology involves the handling of small amounts of liquid in miniaturized systems. In the life sciences, microfluidics usually entails the scaling down of at least one application, such as electrophoresis, to chip format, which often results in increased efficiency and reliability. Bio-COBRA provides a platform for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. Its sensitivity and reproducibility also makes it a tool for the analysis of DNA methylation in clinical samples. The Bio-COBRA assay can be performed on 12 samples in less than 1 h. If the protocol is started at the DNA isolation step, however, approximately 48 h would be required to complete the entire procedure.

A comprehensive search for DNA amplification in lung cancer identifies inhibitors of apoptosis cIAP1 and cIAP2 as candidate oncogenes.

Amplification of oncogenes is an important mechanism that can cause gene overexpression and contributes to tumor development. The identification of amplified regions might have both prognostic and therapeutic significance. We used primary lung carcinomas and lung cancer cell lines for restriction landmark genomic scanning (RLGS) to identify novel amplified sequences. Enhanced RLGS fragments that indicate gene amplification were observed in primary tumors and lung cancer cell lines of both non-small cell lung cancer and small cell lung cancer. We identified one novel amplicon on chromosome 11q22, in addition to previously reported amplicons that include oncogenes MYCC, MYCL1 and previously identified amplification of chromosomal regions 6q21 and 3q26-27. Amplification of 11q22 has been reported in other types of cancer and was refined to an approximately 1.19 Mbp region for which the complete sequence is available. Based on a patient sample with a small region of low-level amplification we were able to further narrow this region to 0.92 Mbp. Genes localized in this region include two inhibitors of apoptosis (cIAP1 and cIAP2). Immunohistochemistry and western blot analysis identified cIAP1 and cIAP2 as potential oncogenes in this region as both are overexpressed in multiple lung cancers with or without higher copy numbers.