Aging in heterozygous Dnmt1-deficient mice: effects on survival, the DNA methylation genes, and the development of amyloidosis

We previously reported that heterozygous DNA methyltransferase 1-deficient (Dnmt1(+/-)) mice maintain T-cell immune function and DNA methylation levels with aging, whereas controls develop autoimmunity, immune senescence, and DNA hypomethylation. We therefore compared survival, cause of death, and T-cell DNA methylation gene expression during aging in Dnmt1(+/-) mice and controls. No difference in longevity was observed, but greater numbers of Dnmt1(+/-) mice developed jejunal apolipoprotein AII amyloidosis. Both groups showed decreased Dnmt1 expression with aging. However, expression of the de novo methyltransferases Dnmt3a and Dnmt3b increased with aging in stimulated T cells from control mice. MeCP2, a methylcytosine binding protein that participates in maintenance DNA methylation, increased with age in Dnmt1(+/-) mice, suggesting a mechanism for the sustained DNA methylation levels. This model thus provides potential mechanisms for DNA methylation changes of aging, and suggests that changes in DNA methylation may contribute to some forms of amyloidosis that develop with aging.     

DNA methylation affects cell proliferation, cortisol secretion and steroidogenic gene expression in human adrenocortical NCI-H295R cells

Aberrant DNA methylation may be involved in human adrenocortical tumorigenesis, which is often accompanied by abnormal hormone production. In this study, we aimed to clarify the effects of DNA methylation on steroidogenesis using the human adrenocortical NCI-H295R cell line as a model. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (Azad; 10 microM for 7 days) decreased the proliferation rate to approximately 20% and the cell number to 60% of the control, with a simultaneous increase in the expression of the cyclin-dependent kinase inhibitor p57(KIP2) gene. In addition, Azad treatment increased cortisol secretion dose and time dependently, whereas dehydroepiandrosterone sulfate secretion was not affected. Azad treatment decreased basal and (Bu)2cAMP-induced expression of low- and high-density lipoprotein receptor, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, steroid 17alpha-hydroxylase/17,20-lyase and steroid 21-hydroxylase mRNA, as well as the StAR protein level. In contrast, Azad treatment increased the basal expression of steroid 11beta-hydroxylase and 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase genes, although it inhibited the (Bu)2cAMP-induced expression of these two genes. The expression of steroidogenic factor-1 (SF-1) and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome 1) genes (both harboring putative CpG islands in their promoters) and the methylation degree of the HpaII recognition site(s) in the SF-1 gene promoter region were reduced by Azad treatment. The immunostaining pattern of the methyl-CpG-binding protein MeCP2 was also modified by Azad treatment. These results suggest that DNA methylation may be implicated in the regulation of cell proliferation and steroidogenesis in human adrenocortical cells.     

DNA methylation analysis techniques

DNA methylation contributes to the control of gene expression and plays an essential role in cellular physiology. Well-defined patterns of DNA methylation are established and fixed during embryonic development, and changes in these patterns may be a contributing factor in developmental disorders, cancer and aging. Not least the possibility of using DNA methylation as a marker for disease has created a strong need for techniques to detect and measure DNA methylation. Different techniques provide information on DNA methylation at different levels, spanning from genome-wide methylation content to methylation of single residues in specific genes. The limitations of individual techniques strongly affect interpretation of data. In this review, we discuss some general themes in DNA methylation analysis and outline the basic principles of current key techniques. We discuss the advantages and disadvantages of these techniques, including potential artifacts and pitfalls, and suggest some overall guidelines that may be instructive for a rational choice of methodology.This revised version was published online in October 2005 with corrections to the Cover Date.     

DNA methylation in inflammatory bowel disease and beyond

Inflammatory bowel disease(IBD)is a consequence of the complex,dysregulated interplay between genetic predisposition,environmental factors,and microbial composition in the intestine.Despite a great advancement in identifying host-susceptibility genes using genome-wide association studies(GWAS),the majority of IBD cases are still underrepresented.The immediate challenge in post-GWAS era is to identify other causative genetic factors of IBD.DNA methylation has received increasing attention for its mechanistical role in IBD pathogenesis.This stable,yet dynamic DNA modification,can directly affect gene expression that have important implications in IBD development.The alterations in DNA methylation associated with IBD are likely to outset as early as embryogenesis all the way until old-age.In this review,we will discuss the recent advancement in understanding how DNA methylation alterations can contribute to the development of IBD.     

The emerging roles of DNA methylation in the clinical management of prostate cancer

Aberrant DNA methylation is one of the hallmarks of carcinogenesis and has been recognized in cancer cells for more than 20 years. The role of DNA methylation in malignant transformation of the prostate has been intensely studied, from its contribution to the early stages of tumour development to the advanced stages of androgen independence. The most significant advances have involved the discovery of numerous targets such as GSTP1, Ras-association domain family 1A (RASSF1A) and retinoic acid receptor beta2 (RARbeta2) that become inactivated through promoter hypermethylation during the course of disease initiation and progression. This has provided the basis for translational research into methylation biomarkers for early detection and prognosis of prostate cancer. Investigations into the causes of these methylation events have yielded little definitive data. Aberrant hypomethylation and how it impacts upon prostate cancer has been less well studied. Herein we discuss the major developments in the fields of prostate cancer and DNA methylation, and how this epigenetic modification can be harnessed to address some of the key issues impeding the successful clinical management of prostate cancer.     

Computational prediction of methylation status in human genomic sequences

Epigenetic effects in mammals depend largely on heritable genomic methylation patterns. We describe a computational pattern recognition method that is used to predict the methylation landscape of human brain DNA. This method can be applied both to CpG islands and to non-CpG island regions. It computes the methylation propensity for an 800-bp region centered on a CpG dinucleotide based on specific sequence features within the region. We tested several classifiers for classification performance, including K means clustering, linear discriminant analysis, logistic regression, and support vector machine. The best performing classifier used the support vector machine approach. Our program (called hdfinder) presently has a prediction accuracy of 86%, as validated with CpG regions for which methylation status has been experimentally determined. Using hdfinder, we have depicted the entire genomic methylation patterns for all 22 human autosomes.     

DNA methylation in liver diseases

Recently, growing evidences show that the combination of epigenetic and genetic abnormalities contribute together to the development of liver diseases. DNA methylation is a very important epigenetic mechanism in human beings. It refers to addition of the methyl groups to DNA and mainly occurs at cytosine adjacent to guanine. DNA methylation is prevalent across human genome and is essential for the normal human development, while its dysfunction is associated with many human diseases. A deep understanding of DNA methylation may provide us deep insight into the origination of liver diseases. Also, it may provide us new tools for diseases diagnosis and prognosis prediction. This review summarized recent progress of DNA methylation study and provided an overview of DNA methylation and liver diseases. Meanwhile, the association between DNA methylation and liver diseases including hepatocellular carcinoma, liver fibrosis, nonalcoholic steatohepatitis and liver failure were extensively discussed. Finally, we discussed the potential of DNA methylationtherapeutics for liver diseases and the value of DNA methylation as biomarkers for liver diseases diagnosis and prognosis prediction. This review aimed to provide the emerging DNA methylation information about liver diseases. It might provide essential information for managing and care of these patients.     

Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a

Current evidence indicates that methylation of cytosine in mammalian DNA is restricted to both strands of the symmetrical sequence CpG, although there have been sporadic reports that sequences other than CpG may also be methylated. We have used a dual-labeling nearest neighbor technique and bisulphite genomic sequencing methods to investigate the nearest neighbors of 5-methylcytosine residues in mammalian DNA. We find that embryonic stem cells, but not somatic tissues, have significant cytosine-5 methylation at CpA and, to a lesser extent, at CpT. As the expression of the de novo methyltransferase Dnmt3a correlates well with the presence of non-CpG methylation, we asked whether Dnmt3a might be responsible for this modification. Analysis of genomic methylation in transgenic Drosophila expressing Dnmt3a reveals that Dnmt3a is predominantly a CpG methylase but also is able to induce methylation at CpA and at CpT.