DNA methylation pattern in a barley reconstructed karyotype with deleted ribosomal gene cluster of chromosome 6H

A reconstructed barley karyotype (T-35) was utilised to study the influence of chromosomal rearrangements on the DNA methylation pattern at chromosome level. Data obtained were also compared with the distribution of Giemsa N-bands and high gene density regions along the individual chromosomes that have been previously described. In comparison to the control karyotype (T-1586), the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complement. Significant remodelling process of methylation pattern was found also in the residual nucleolus organiser regions (NOR) on chromosome 5H as a consequence of deletion comprising the whole NOR of chromosome 6H in T-35. Moreover, differences between corresponding segments of the homologues with respect to some other chromosome locations were also observed. Repositioning of genomic DNA methylation along the metaphase chromosomes following chromosomal reconstruction in barley seems to be essential to ensure correct chromatin organisation and function.     

Methylation and expression of the human thyroglobulin gene

The DNA methylation pattern at the 5'end of the human thyroglobulin gene has been determined in different tissues. Out of the four HpaII/MspI sites (5'-CCGG-3') present in this region, three were found to be non-methylated in thyroid DNA, while full methylation was observed in liver, salivary gland and sperm DNA. This demethylation therefore correlates with expression of the thyroglobulin gene. However, all four sites were found to be non-methylated in placental DNA, regardless of the activity of the gene.     

DNA methylation changes in human testicular cancer

We previously reported an X/Y imbalance with a relative excess of X- and a relative deficiency of Y-chromosomal DNA in three out of nine testicular tumors of germ cell origin. To study the implications of those changes the methylation status of DNA from seven of the tumors was explored by HpaII/MspI analysis. The 5' regions of the hypoxanthine phosphoribosyltransferase (HPRT) and the phosphoglycerate kinase (PGK) gene loci exhibited main patterns suggestive of active X chromosomes in the tumors. However, a minority of the HPRT loci of one teratocarcinoma with an increased dosage of the X chromosome, as well as one additional teratocarcinoma, revealed patterns analogous to inactive X chromosomes in females. Using probes from several chromosomes it was subsequently found that the teratocarcinoma tumors (3/3) were characterized by generalized hypermethylation. On the contrary, the seminomas showed variable hypomethylation (4/5) or virtually complete demethylation (1/5). The seminoma with the most extensive hypomethylation was disseminated (stage III), whereas the other seminomas were local (stage I). These findings suggest that DNA methylation may play a role in the developmental pathways leading to different histologic types of testicular tumors of germ cell origin. The HPRT results imply that the consequences of extra X chromosomes--a frequent finding in testicular tumors--may be modulated by mechanisms, such as DNA methylation, that control gene activity.     

Reproducibility and concordance of differential DNA methylation and gene expression in cancer

Background

Hundreds of genes with differential DNA methylation of promoters have been identified for various cancers. However, the reproducibility of differential DNA methylation discoveries for cancer and the relationship between DNA methylation and aberrant gene expression have not been systematically analysed.

Methodology/Principal Findings

Using array data for seven types of cancers, we first evaluated the effects of experimental batches on differential DNA methylation detection. Second, we compared the directions of DNA methylation changes detected from different datasets for the same cancer. Third, we evaluated the concordance between methylation and gene expression changes. Finally, we compared DNA methylation changes in different cancers. For a given cancer, the directions of methylation and expression changes detected from different datasets, excluding potential batch effects, were highly consistent. In different cancers, DNA hypermethylation was highly inversely correlated with the down-regulation of gene expression, whereas hypomethylation was only weakly correlated with the up-regulation of genes. Finally, we found that genes commonly hypomethylated in different cancers primarily performed functions associated with chronic inflammation, such as ‘keratinization’, ‘chemotaxis’ and ‘immune response’.

Conclusions

Batch effects could greatly affect the discovery of DNA methylation biomarkers. For a particular cancer, both differential DNA methylation and gene expression can be reproducibly detected from different studies with no batch effects. While DNA hypermethylation is significantly linked to gene down-regulation, hypomethylation is only weakly correlated with gene up-regulation and is likely to be linked to chronic inflammation.     

Methylation of an alpha-foetoprotein gene intragenic site modulates gene activity.

By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding.     

Absence of expression of the FMR-1 gene in fragile X syndrome

We previously reported the isolation of a gene (FMR-1) expressed in brain at the fragile X locus. One exon of this gene lies within an EcoRI fragment that exhibits length variation in fragile X patients. This exon also contains the CGG repeat within the CpG island hypermethylated in fragile X patients. To study the involvement of the FMR-1 gene in the fragile X syndrome, its expression was studied in lymphoblastoid cell lines and leukocytes derived from patients and normal controls. FMR-1 mRNA was absent in the majority of male fragile X patients, suggesting a close involvement of this gene in development of the syndrome. Normal individuals and carriers all show expression. The methylation status of the BssHII site at the CpG island was also studied by Southern blot analysis of DNA from patients, carriers, and controls. The minority of fragile X affected males that show expression of FMR-1 demonstrated an associated incomplete methylation of the BssHII site.     

Differential methylation of the c-H-ras gene in normal mouse cells and during skin tumour progression.

We have previously shown that the mouse c-H-ras gene acquires transforming activity in chemically induced skin tumours. We have now investigated the pattern of DNA methylation at HpaII and XhoI sites around the c-H-ras locus in various tissues and stages of epidermal tumour progression. The results of this study suggest a correlation between the methylation state of the c-H-ras gene and its susceptibility to oncogenic conversion by a point-mutation. The locus is substantially undermethylated in normal epidermis in comparison with NIH/3T3 fibroblasts. Intermediate levels of methylation were observed in the other tissues investigated. The undermethylation at HpaII sites in epidermal DNA persists through the morphologically distinct phases of hyperplasia, benign papilloma and malignant carcinoma. Methylation at a specific XhoI site close to the c-H-ras gene is significantly reduced with respect to normal epidermis in some, but not all epidermal tumours. The methylation state of the c-H-ras locus in specific tumours is stably maintained following transfection of these DNAs into NIH/3T3 cells and selection of transformed foci. Demethylation of the locus is not essential in vitro for the transforming activity of DNA from epidermal tumours. The significance of changes in the methylation pattern of the c-H-ras gene in different tissues and during tumour progression is discussed.     

DNA methylation changes in cell line from beta-lactoglobulin gene targeted fetus

The gene targeting combined somatic cell nuclear transfer is very useful in agriculture and medicine. Epigenetic modification of DNA by methylation is significant in regulating gene expression during mammalian development. During gene targeting, epigenetic status of donor cell nuclei may be changed in a series of processes, including homologous recombination, cell selection and cloning. We examined DNA methylation of six genes (beta-actin, VEGF, oct4, TERT, H19 and Igf2) and a repetitive sequence art2 in blg(+/-) cell line from beta-lactoglobulin (BLG) gene targeted fetus and the cells used for BLG gene targeting serve as control. The results demonstrated that the widespread changes of DNA methylation were found in blg(+/-) cell line. But the degree of variation was different. DNA methylation of VEGF in blg(+/-) was noticeably decreased. These observations suggest that DNA methylation variations may impact gene expression and finally induce abnormalities and lethality in later developmental stages.