Genome-Wide Transcriptome and Binding Sites Analyses Identify Early FOX Expressions for Enhancing Cardiomyogenesis Efficiency of hESC Cultures.

The differentiation efficiency of human embryonic stem cells (hESCs) into heart muscle cells (cardiomyocytes) is highly sensitive to culture conditions. To elucidate the regulatory mechanisms involved, we investigated hESCs grown on three distinct culture platforms: feeder-free Matrigel, mouse embryonic fibroblast feeders, and Matrigel replated on feeders. At the outset, we profiled and quantified their differentiation efficiency, transcriptome, transcription factor binding sites and DNA-methylation.

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups.

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.

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.

Bio-COBRA: absolute quantification of DNA methylation in electrofluidics chips.

DNA methylation is the best-studied epigenetic modification, and in mammals it describes the conversion of cytosine to 5-methylcytosine in the context of CpG dinucleotides. In recent years, it has become evident that epigenetic mechanisms are severely disrupted in human neoplasia, and evidence suggests that alterations of DNA methylation patterns may be an integral mechanism in the etiology of other diseases such as bipolar disorder and schizophrenia. The main effect of altered DNA methylation is the disruption of normal patterns of gene expression through genomic instability and hypermethylation of CpG islands, which together could lead to uncontrolled cell proliferation.

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.

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.

Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome.

Widespread provision of human embryonic stem cells (hESCs) for therapeutic use, drug screening and disease modelling will require cell lines sustainable over long periods in culture. Since the short-term, in vitro culture of mammalian embryos can result in DNA methylation changes, the epigenetic stability of hESCs warrants investigation. Existing hESC lines have been derived and cultured under diverse conditions, providing the potential for programming differential changes into the epigenome that may result in inter-line variability over and above that inherited from the embryo.

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 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.

Mining methylation for early detection of common cancers.

A single method that detects multiple common cancer types at an early stage would have the biggest payoff for cancer control, say Brena and colleagues.

CpG island hypermethylation profiling of lung cancer using restriction landmark genomic scanning (RLGS) analysis.

Lung cancer remains the leading cause of cancer related mortality, accounting for almost one-third of cancer deaths in men and one-fourth of cancer deaths in women; 160,440 lung cancer deaths are expected in 2004. Survival from lung cancer depends mainly upon the stage at presentation. As localized tumors generally do not cause symptoms, the disease is usually diagnosed in symptomatic patients at advanced stages when the prognosis is poor.

Epigenetic modulation of tumor suppressor CCAAT/enhancer binding protein alpha activity in lung cancer.

Background: Loss of tumor suppressor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) expression is seen in several human malignancies, including acute myelogenous leukemia and lung cancer. We hypothesized that DNA methylation and histone acetylation of the C/EBPalpha promoter may modulate C/EBPalpha expression in lung cancer.

Methods: We analyzed C/EBPalpha expression in 15 human lung cancer cell lines and in 122 human lung primary tumors by northern blotting, immunoblotting, and immunohistochemistry.

Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform.

DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation.

Quantitative assessment of DNA methylation: Potential applications for disease diagnosis, classification, and prognosis in clinical settings.

Deregulation of the epigenome is now recognized as a major mechanism involved in the development and progression of human diseases such as cancer. As opposed to the irreversible nature of genetic events, which introduce changes in the primary DNA sequence, epigenetic modifications are reversible and leave the original DNA sequence intact. There is now evidence that the epigenetic landscape in humans undergoes modifications as the result of normal aging, with older individuals exhibiting higher levels of promoter hypermethylation compared to younger ones.

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.

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.