Genomic DNA methylation in health and disease

University dissertation from Stockholm : Karolinska Institutet, Department of Molecular Medicine and Surgery

Abstract: Cytosine methylation is one of the important epigenetic mechanisms controlling the vertebrate genome. Changes in genomic DNA methylation have been demonstrated in aging, auto-immune diseases and cancer. Aberrant DNA methylation patterns, involving both hypomethylation and hypermethylation, are associated with pathological events in cancer and other diseases. The aim of this thesis was to shed light on DNA methylation and its control in disease. Initially, a novel assay to estimate global DNA methylation was developed which was named LUminometric Methylation Assay (LUMA). In this assay, DNA cleavage by methylation-sensitive restriction enzymes is coupled to a polymerase extension assay by Pyrosequencing . LUMA is a quantitative, highly reproducible and easy to scale up assay which can be applied as a useful method to analyze genome-wide DNA methylation in a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer. In the next step of the study, it was shown, for the first time, that global DNA hypermethylation is correlated with systemic inflammation in chronic kidney disease patients. Moreover, it was demonstrated that there is an association between global DNA hypermethylation and survival rate in end stage renal patients starting dialysis treatment. A correlation between genomic DNA hypermethylation and cardiovascular disease was revealed in this study. It was suggested that DNA methylation can be used as a strong novel prognostic marker in these renal patients. Changes in DNA methylation by TrichostatinA (TSA) treatment was investigated in Hep3B cells as part of the study. To address this, the effect of TSA on DNA methylation was studied at gene specific and global levels. It was shown that TSA treatment results in genomic hypomethylation by affecting DNA methyltransferases both at the protein level and by changes in the DNA methyltransferases (DNMTs) mobility in the nucleus. Finally, the impact of cytomegalovirus (CMV) infection on the host cell DNA methylation machinery was investigated. Using several experimental approaches it was shown that CMV infection alters the DNA-methylation machinery of the host cell leading to profound global inhibition of DNA methylation by intracellular relocalization of DNMT1. It was also suggested that defects in the DNMT1 function render non-permissive cells to be permissive to CMV infection. These findings may also help to understand the etiology of CMV pathogenesis and develop new therapy. Based on the data achieved during this study, it has been suggested that cellular DNA methylation is a dynamic process which is changed based on cellular responses to physiological environment.

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