Genome DNA Methylation Analysis Research Articles

Lentiviral Transduction of CD34+ Cells Induces Genome-Wide Epigenetic Modifications

Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols. In an experimental model of ex vivo gene modification for hematopoietic gene therapy, human CD34+ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34+ cells to cytokines increased nuclear expression of epigenetic regulators SIRT1 or DNMT1 and caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34+ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.

In hepatocellular carcinoma miR‐519d is up‐regulated by p53 and DNA hypomethylation and targets CDKN1A/p21, PTEN, AKT3 and TIMP2

MiR-519d belongs to the chromosome 19 miRNA cluster (C19MC), the largest human miRNA cluster. One of its members, miR-519d, is over-expressed in hepatocellular carcinoma (HCC) and we characterized its contribution to hepatocarcinogenesis. In HCC cells, the over-expression of miR-519d promotes cell proliferation, invasion and impairs apoptosis following anticancer treatments. These functions are, at least in part, exerted through the direct targeting of CDKN1A/p21, PTEN, AKT3 and TIMP2. The mechanisms underlying miR-519d aberrant expression in HCC were assayed by genomic DNA amplification, methylation analysis and ChIP assay. The aberrant hypomethylation of C19MC and TP53 were respectively identified as an epigenetic change allowing the aberrant expression of miR-519d and one of the factors able to activate its transcription. In conclusion, we assessed the oncogenic role of miR-519d in HCC by characterizing its biological functions, including the modulation of response to anticancer treatments and by identifying CDKN1A/p21, PTEN, AKT3 and TIMP2 among its targets.

A Distinct DNA Methylation Pattern Characterizes Childhood Acute Lymphoblastic Leukemia and Separates Patients Into Prognostic Groups,

Abstract 3568Acute lymphoblastic Leukemia (ALL) is the most common malignancy in children. Risk stratification at initial diagnosis is essential for an intensity adapted therapy.During the last years epigenetic processes such as DNA methylation have been discovered to play an important role in the pathogenesis of leukemia. Nevertheless up to now there is only little information whether deregulation of DNA methylation has a prognostic impact and can be associated with the response to therapy and outcome of patients. However, identification of such prognostic methylation markers might improve risk prediction and individualized therapy approaches.In order to identify ALL-specific DNA methylation pattern as well as risk-associated aberrant DNA methylation we analyzed a cohort of 100 ALL samples (79 B-ALL, 21 T-ALL) of the ALL-BFM 2000 study including 36 matched remissions. Classification into risk groups was based on prednisone response and positivity for BCR/ABL or MLL/AF4 fusion transcripts as well as minimal residual disease. For whole genome DNA methylation analysis we used the newly developed Infinium HumanMethylation450 BeadChip covering 482,421 CpG dinucleotides of the human Genome.Unsupervised hierarchical clustering showed that the ALL samples, independent of immunphenotype or risk group, clustered separately from the remission samples. A total of 18,326 promoter CpGs referring to 3491 genes were differentially methylated in ALL compared with remission controls. About 95% of these genes were hypermethylated by more than 2-fold (p<0.001). Genes predominantly affected by differential methylation were associated with Cellular and Tissue Development and Cell Signaling. Notably, several genes of the PAX- and SIX-families, comprising transcription factors that regulate proliferation and development, were hypermethylated in the ALL samples. A correlation with microarray-RNA-expression data comparing ALL vs. healthy (www.leukemia-gene-atlas.org) showed an inverse correlation between DNA methylation and mRNA levels for about half of the top 50 hypermethylated genes and of 70% of the top 50 hypomethylated genes.Interestingly, hierarchical clustering revealed that patients grouped into high risk and intermediate risk clustered apart from patients of the standard risk group. Comparing high risk (HR) and standard risk (SR) groups, 860 differentially methylated genes could be detected and the majority (approximately 80%) was hypermethylated in the HR group. Hypermethylated genes predominantly functioned in gene expression- (e.g. HDAC9, HDAC4 and DNMT3A), cell death- as well as hematological development-pathways. Hypomethylated genes mainly belonged to cellular growth and proliferation- as well as signaling pathways. Interestingly, the ABC transporter ABCC4 that confers drug resistance in solid tumors was also hypomethylated in the HR group. Prednisone response was also associated with DNA methylation differences: A total of 325 genes were differentially methylated between good and poor prednisone responders. Again, ABCC4 was hypomethylated in the poor-response group potentially indicating increased drug resistance. Correlation of differentially methylated genes in the HR- and poor-prognostic groups showed an overlap in 293 genes, probably representing the gene set whose aberrant methylation might be associated with therapy failure.Taken together, genome spanning methylation analyses can identify prognosis and therapy response associated markers. Some of these methylation changes might also be functionally associated with therapy resistance. Disclosures:No relevant conflicts of interest to declare.

Advances in Genomic DNA Methylation Analysis

Advances in Genomic DNA Methylation Analysis

Genes Associated with Recurrence of Hepatocellular Carcinoma: Integrated Analysis by Gene Expression and Methylation Profiling

Gene expression is suppressed by DNA methylation. The goal of this study was to identify genes whose CpG site methylation and mRNA expression are associated with recurrence after surgical resection for hepatocellular carcinoma (HCC). Sixty-two HCCs were examined by both whole genome DNA methylation and transcriptome analysis. The Cox model was used to select genes associated with recurrence. A validation was performed in an independent cohort of 66 HCC patients. Among fifty-nine common genes, increased CpG site methylation and decreased mRNA expression were associated with recurrence for 12 genes (Group A), whereas decreased CpG site methylation and increased mRNA expression were associated with recurrence for 25 genes (Group B). The remaining 22 genes were defined as Group C. Complement factor H (CFH) and myosin VIIA and Rab interacting protein (MYRIP) in Group A; proline/serine-rich coiled-coil 1 (PSRC1), meiotic recombination 11 homolog A (MRE11A), and myosin IE (MYO1E) in Group B; and autophagy-related protein LC3 A (MAP1LC3A), and NADH dehydrogenase 1 alpha subcomplex assembly factor 1 (NDUFAF1) in Group C were validated. In conclusion, potential tumor suppressor (CFH, MYRIP) and oncogenes (PSRC1, MRE11A, MYO1E) in HCC are reported. The regulation of individual genes by methylation in hepatocarcinogenesis needs to be validated.

Advances in Genomic DNA Methylation Analysis

Advances in Genomic DNA Methylation Analysis

Whole genome DNA methylation analysis based on high throughput sequencing technology

There are numerous approaches to decipher a whole genome DNA methylation profile (“methylome”), each varying in cost, throughput and resolution. The gold standard of these methods, whole genome bisulfite-sequencing (BS-seq), involves treatment of DNA with sodium bisulfite combined with subsequent high throughput sequencing. Using BS-seq, we generated a single-base-resolution methylome in human peripheral blood mononuclear cells (in press). This BS-seq map was then used as the reference methylome to compare two alternative sequencing-based methylome assays (performed on the same donor of PBMCs): methylated DNA immunoprecipitation (MeDIP-seq) and methyl-binding protein (MBD-seq). In our analysis, we found that MeDIP-seq and MBD-seq are complementary strategies, with MeDIP-seq more sensitive to highly methylated, high-CpG densities and MDB-seq more sensitive to highly methylated, moderate-CpG densities. Taking into account the size of a mammalian genome and the current expense of sequencing, we feel 3gigabases (Gbp) 45bp paired-end MeDIP-seq or MBD-seq uniquely mapped reads is the minimum requirement and cost-effective strategy for methylome pattern analysis.

Genetic Mechanisms and Aberrant Gene Expression during the Development of Gastric Intestinal Metaplasia and Adenocarcinoma

Gastric adenocarcinoma occurs via a sequence of molecular events known as the Correa’s Cascade which often progresses over many years. Gastritis, typically caused by infection with the bacterium H. pylori, is the first step of the cascade that results in gastric cancer; however, not all cases of gastritis progress along this carcinogenic route. Despite recent antibiotic intervention of H. pylori infections, gastric adenocarcinoma remains the second most common cause of cancer deaths worldwide. Intestinal metaplasia is the next step along the carcinogenic sequence after gastritis and is considered to be a precursor lesion for gastric cancer; however, not all patients with intestinal metaplasia develop adenocarcinoma and little is known about the molecular and genetic events that trigger the progression of intestinal metaplasia into adenocarcinoma. This review aims to highlight the progress to date in the genetic events involved in intestinal-type gastric adenocarcinoma and its precursor lesion, intestinal metaplasia. The use of technologies such as whole genome microarray analysis, immunohistochemical analysis and DNA methylation analysis has allowed an insight into some of the events which occur in intestinal metaplasia and may be involved in carcinogenesis. There is still much that is yet to be discovered surrounding the development of this lesion and how, in many cases, it develops into a state of malignancy.

LUMA (LUminometric Methylation Assay)—A high throughput method to the analysis of genomic DNA methylation

Changes in genomic DNA methylation are recognized as important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as cancer and other diseases. Here, we report a novel method to estimate genome-wide DNA methylation, referred to as LUminometric Methylation Assay (LUMA). The method is based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by Pyrosequencing™. The method is quantitative, highly reproducible and easy to scale up. Since no primary modification of genomic DNA, such as bisulfite treatment, is needed, the total assay time is only 6 h. In addition, the assay requires only 200–500 ng of genomic DNA and incorporates an internal control to eliminate the problem of varying amounts of starting DNA. The accuracy and linearity of LUMA were verified by in vitro methylated lambda DNA. In addition, DNA methylation levels were assessed by LUMA in DNA methyltransferase knock-out cell lines and after treatment with the DNA methyltransferase inhibitor (5-AzaCytidine). The LUMA assay may provide a useful method to analyze genome-wide DNA methylation for a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer.

Specific Method for the Determination of Genomic DNA Methylation by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

Herein we report a novel method for determining genomic DNA methylation that utilizes liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2'-deoxycytidine levels following enzymatic hydrolysis of genomic DNA. LC separation of 5-methyl-2'-deoxycytidine from the four deoxyribonucleosides, the four ribonucleosides, and 5-methyl-2'-cytidine, a RNA methylation product, has been achieved within 15 min. In combination with ESI-MS/MS detection, the reported method is highly specific and extremely sensitive with a limit of detection (LOD) of 0.2 fmol and a quantification linearity range from 1 fmol to 20 pmol. Genomic DNA methylation was expressed as the ratio of 5-methyl-2'-deoxycytidine to 2'-deoxyguanosine and was determined directly using 2'-deoxyguanosine as the internal standard. Because deoxycytidine methylation typically ranges from 2 to 6% in mammalian genomes, and pharmacological or genetic manipulations have not achieved levels lower than 0.1%, we validated the assay for methylation levels ranging from 0.05 to 10%. Importantly, both RNA contamination and incomplete DNA hydrolysis had no appreciable effect on 5-methyl-2'-deoxycytidine quantification. LOD studies indicate that only 4 ng of DNA is required for this assay. This LOD should permit the use of this method for applications having limiting amounts of DNA that were not previously candidates for global genomic DNA methylation analysis, e.g., clinical trial samples, or cells collected by laser capture microdissection.

Novel methods for analysis of genomic DNA methylation

Novel methods for analysis of genomic DNA methylation

Denaturing gradient gel electrophoresis to detect methylation changes in DNA.

Denaturing gradient gel electrophoresis (DGGE) is a technique that fractionates DNA molecules on the basis of their melting behavior and thereby permits the separation of DNA fragments with local variations in base composition. The separation of DNA fragments by DGGE is determined by the nucleotide sequence, rather than size. This approach is effective when part of the molecule is relatively dense in G+C pairs. This separation is possible because of the pronounced drop in electrophoretic mobility in a polyacrylamide gel that occurs when a region of a DNA molecule melts, thereby forming a structure that is partly helical and partly random chain. The electrophoretic mobility of these partly melted DNA fragments is much lower than that of fully helical or fully dissociated molecules. The low residual mobility of the fragment restricts migration into more strongly denaturing regions of the gradient gel and results in focusing of the band. This property can be applied to detect the difference in melting temperature between methylated and nonmethylated DNA fragments after chemical treatment, or to enrich genomic regions in which aberrant methylation occurs. In this chapter, the application of DGGE to the analysis of genomic DNA methylation is reviewed.

Natural and conditional ageing of mouse fibroblasts: Genetic vs. epigenetic control

Mouse embryonic fibroblasts were fused with a spontaneously immortalized mouse fibroblast cell line and a large number of heterogeneous hybrid populations were obtained. These showed variations with respect to their chromosomal number as well as in vitro life span but none aquired immortal phenotype. Apart from demonstrating the dominant nature of senescence over immortalization in mouse system, we also provide the first report on the analysis of genomic DNA methylation during in vitro passaging of parental and hybrid cell populations representing the normal and conditional ageing, respectively. Since no random decline in DNA methylation could be detected in any of the cases, our results suggest that it is unlikely that mortality of cells in culture is the outcome of random loss of epigenetic control imposed by 5-methyldeoxycytidine at CpG sites in the genome.