Functional Analysis of DNA Methylation in Development and Disease

Gaudet, François (2006): Functional Analysis of DNA Methylation in Development and Disease. Dissertation, LMU München: Fakultät für Biologie

Abstract

The genome of mammals harbors chemical modifications at some cytosine residues in the form of a methyl group. These modified residues, termed 5’-methylcytosines, have been discovered more than 50 years ago (Hotchkiss 1948) and have since been shown to play important roles in the regulation of gene expression and in the execution of developmental programs. Patterns of cytosine methylation (also referred to as DNA methylation) are carefully set and preserved during cellular expansion and global methylation levels are well regulated throughout development. Changes in methylation patterns and levels have been associated with disease progression and death (Li et al. 1992; Okano et al. 1999; Ehrlich 2002). Specifically, elevated levels of global genomic methylation have been shown to play a role in the inactivation of tumor suppressor genes in many types of cancer (Ehrlich 2002). In contrast, reduced levels of methylation have been observed in a wide variety of tumors and complete demethylation in vivo causes embryonic death (Li et al. 1992; Ehrlich 2002). In an effort to study the effect of changed methylation levels in vivo and its effect on disease progression, we developed a genetic approach to study the effect of hypomethylation during embryogenesis and adulthood. DNA methyltransferase 1 (Dnmt1) is the major methyltransferase in mammals and genetic inactivation of the Dnmt1 gene causes demethylation that results in cell death in tissue culture and embryonic lethality of homozygous mutant mice at E8.5 (Li et al. 1992). In a first step, the 5’ end of the Dnmt1 gene was characterized to determine the structure of a new oocyte-specific isoform found in oocytes and early embryos. Upon elucidation of the structure of this isoform, assays were developed to test its function in vivo. Loss of this oocyte-specific isoform protein resulted in hypomethylation of an IAP reporter element suggesting a role for this protein in early development. In contrast, the somatic Dnmt1 isoform, which is present in all somatic cells, was important for maintaining this IAP element methylated following implantation of the embryo and throughout adulthood. Reduced levels of Dnmt1 in adults caused global hypomethylation and resulted in the development of thymic lymphomas which displayed a duplication of chromosome 15 (trisomic 15). The c-myc oncogene, which resides on chromosome 15, was overexpressed, and a gene expression array analysis revealed that another oncogene, Notch-1, was also overexpressed in all tumors. Cooperation between those oncogenes has been previously shown to induce thymic lymphomas. Analysis of the Notch-1 locus demonstrated the presence of IAP insertions upstream of the oncogenic cytoplasmic domain capable of activating transcription of truncated oncogenic Notch-1. IAP elements were shown to be activated by hypomethylation albeit not as much as traditional mutagenic retroviruses. These results thus show that hypomethylation may induce tumorigenesis in this model following two mechanisms. First by inducing chromosome instability and second by creating insertional mutagenesis of defective retroviral elements such as IAPs. These results demonstrate for the first time that hypomethylation can directly induce tumorigenesis in mice and induce chromosome instability.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: epigenetics, DNA methylation, cancer, DNA hypomethylation, Dnmt1
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie ; 500 Naturwissenschaften und Mathematik
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 30. Mai 2006
• 1. Bericht­erstatter:in: Leonhardt, Heinrich