Methylation-sensitive Single Nucleotide Primer Research Articles

Epigenetic regulation of microRNAs in cancer: An integrated review of literature

MicroRNAs (miRNAs) belong to the heterogeneous class of non-coding RNAs (ncRNAs) that regulate the translation and degradation of target mRNAs, and control approximately 30% of human genes. MiRNA genes might be silenced in human tumors (oncomiRs) by aberrant hypermethylation of CpG islands that encompass or lie adjacent to miRNA genes and/or by histone modifications. We performed literature search for research articles describing epigenetically regulated miRNAs in cancer and identified 45 studies that were published between 2006 and 7/2010. The data from those papers are fragmented and methodologically heterogeneous and our work represents first systematic review towards to integration of diverse sets of information. We reviewed the methods used for detection of miRNA epigenetic regulation, which comprise bisulfite genomic sequencing PCR (BSP), bisulfite pyrosequencing, methylation specific PCR (MSP), combined bisulfite restriction analysis (COBRA), methylation sensitive single nucleotide primer extension (Ms-SNuPE), MassARRAY technique and some modifications of those methods. This integrative study revealed 122 miRNAs that were reported to be epigenetically regulated in 23 cancer types. Compared to protein coding genes, human oncomiRs showed an order of magnitude higher methylation frequency (11.6%; 122/1048 known miRNAs). Nearly half, (45%; 55/122) epigenetically regulated miRNAs were associated with different cancer types, but other 55% (67/122) miRNAs were present in only one cancer type and therefore representing cancer-specific biomarker potential. The data integration revealed miRNA epigenomic hot spots on the chromosomes 1q, 7q, 11q, 14q and 19q. CpG island analysis of corresponding miRNA precursors (pre-miRNAs) revealed that 20% (26/133) of epigenetically regulated miRNAs had a CpG island within the range of 5 kb upstream, among them 14% (19/133) of miRNAs resided within the CpG island. Our integrative survey and analyses revealed candidate cancer-specific miRNA epigenetic signatures which provide the basis for new therapeutic strategies in cancer by targeting the epigenetic regulation of miRNAs.

Developmentally Regulated Demethylase Activity Targeting the βA-Globin Gene in Primary Avian Erythroid Cells

Differential expression of globin genes has provided an interesting model system for better understanding commonly inherited diseases such as thalassemia. In the avian beta-type globin cluster (5'-rho-betaH-betaA-epsilon-3'), silencing of the embryonic rho-globin gene occurs concomitantly with the activation of the adult betaA-globin gene during embryonic development. DNA methylation is a dynamic process that regulates gene expression. We observed a progressive loss of methylation of betaA-globin gene, during avian embryonic development that was concurrent with the expression of the gene. The promoter and exon 1 regions of the template strand were completely demethylated, whereas residual methylation was retained in exons 2 and 3. Using a modified methylation-sensitive single-nucleotide primer extension (MS-SNuPE) assay, we observed stage-specific demethylase activity in the nuclear extracts of chicken red cells; activity in 5-, 8-, and 11-day-old erythroid cell nuclear extracts was 6, 76, and 24%, respectively. The demethylase targeted both hemimethylated and fully methylated substrates. Our findings demonstrate stage-specific demethylase activity in nuclear extracts from primary chicken erythroid cells that could target the fully methylated promoter of a developmentally regulated native gene.

DNA Methylation Analysis as a Tool for Cell Typing

Cell therapeutic approaches currently lack definitive quality control measures which guarantee safety in clinical applications and create consistent standards for regulatory approval. These approaches rely on isolation, purification and possibly ex-vivo manipulation of donor cells. Since such cells are exposed to artificial environments, there is potential for deviations from natural growth processes. The resulting heterogeneity of cell cultures is an inherent problem. Therefore, verification of cell identity and quantification of subpopulations is mandatory. Focusing on cultured human primary cells, we tested whether DNA-methylation patterns serve as distinctive cell-type markers. We identified panels of cell type-specific differentially methylated gene regions (CDMs) which produce unambiguous profiles for these cell types. Applying methylation sensitive single nucleotide primer extension generated binary cell-type descriptors (“barcodes”) which allow quantification of cell mixtures. Thus, methylation based analytics suggest themselves aspromising tools for the characterisation and quality control of ex-vivo manipulated cells.

Demethylation of βA-Globin Gene in Avian Primary Erythroid Cells Is Developmentally Regulated, Strand Asymmetrical and an Active Process.

Differential expression of globin genes has provided an interesting model system for the better understanding of commonly inherited diseases such as thalassemia. In the avian β-type globin cluster (5′- ρ-βA- βA - ε - 3′), silencing of the embryonic ρ-globin gene occurs concomitantly with the activation of adult βA-globin gene during the embryonic development. The pattern of cytosine methylation plays an important role in gene regulation. We have previously shown that de novo methylation of the embryonic ρ-globin gene during development is strand-asymmetrical and spreads from the proximal transcribed region in the early definitive chicken erythroid cells. In the present study, we examined the methylation pattern of βA- globin gene during development in primary chicken erythroid cells. Using bisulfite genomic sequencing we observed a progressive demethylation of βA- globin gene during embryonic development. Maximum methylation is seen in the different regions of the βA- globin gene on day 5, which reduces on day 8 by about 50 %, and on day 11 the degree of methylation is minimal. Further, strand and regional asymmetry was seen with respect to loss of methylation. The template strand gets demethylated first and demethylation of the coding strand lags behind. The promoter and exon 1 get completely demethylated by day 11 whereas residual methylation is retained on exons 2 and 3. To investigate the mechanism of loss of methylation, we examined the methylation pattern of multiple DNA strands derived from primitive and early definitive embryonic erythroid cells. The primitive embryonic erythroid cells demonstrated methylation of the βA- globin promoter and exon 1 on most of the CpG dinucleotides. Discontinuous loss of methylation in early definitive erythroid cells suggested an active mechanism of demethylation since the DNA replication mediated passive demethylation is likely to be continuous. To investigate if an active process contributes to the demethylation of βA- globin gene, we used a modified methylation sensitive single nucleotide primer extension assay (MS-SNuPE) assay for quantitation of demethylase activity. Demethylase assay was carried out using synthetic unmethylated, hemimethylated, and completely methylated oligonucleotides derived from the βA-globin promoter region and nuclear extracts from embryonic chicken erythroid cells. The demethylase activity in day 5, 8 and 11 nuclear extracts was 6%, 76%, and 24% respectively. In contrast to the previously reported 5-methylcytosine glycosylase, which shows a significant substrate specificity for the hemimethylated DNA, we observed the demethylase activity in the embryonic chicken erythroid cells on both fully methylated and hemimethylated βA-globin promoter sequences. Since human and chicken methyl binding domain 4 (MBD4) proteins exhibit 5-methylcytosine glycosylase activity, we determined the in vivo binding of MBD4 to βA- globin promoter in chicken erythroid cells using Chromatin ImmunoPrecipitation (ChIP) followed by real time polymerase chain reaction. MBD4 binding to βA- globin promoter was seen in day 8 but not day 5 chicken erythroid cells implicating a possible role of chicken MBD4 in mediating the active demethylation of the avian βA- globin gene. Our findings demonstrate that demethylation of βA globin gene in developing primary chicken erythroid cells shows strand and regional asymmetry and occurs at least in part by an active process.

Reduction of p53 gene expression in human primary hepatocellular carcinoma is associated with promoter region methylation without coding region mutation

Functional inactivation of tumor suppressor genes during tumor progression has been shown to occur by either coding region mutation or promoter region methylation. Because of the functional equivalence of these two mechanisms, loss of tumor suppressor function generally occurs by one or the other mechanism, but rarely by both. Aberrant de novo methylation in most tumor suppressor promoter regions is found within CpG islands that occur near the transcription start site. The p53 promoter region is unique in that it does not contain a CpG island and therefore it is possible that methylation at critical CpG sites may be more important in gene silencing than total CpG methylation density. Other than site-specific aflatoxin B 1-induced mutations, p53 coding region mutations are not frequently observed in most human primary hepatocellular carcinomas. In the present study, paired samples of human primary liver carcinoma and uninvolved tissue obtained from the same individual were evaluated for site-specific p53 promoter methylation status by methylation sensitive single nucleotide primer extension (Ms-SNuPE) and also for coding region mutations using polymerase chain reaction (PCR)- single strand conformation polymorphism (SSCP). The methylation pattern in the uninvolved tissue was variable at specific CpG sites, whereas the same sites had become highly methylated in tumor tissue from the same individual. Associated with de novo methylation, the level of p53 mRNA was significantly reduced in the tumor DNA relative to the uninvolved tissue DNA. None of the samples exhibited coding region mutations. Given that p53 mutations are rare in primary human liver tumors, these data suggest that transcriptional repression by p53 promoter methylation may contribute to tumor progression.