Functional Analysis of DNA Methylation in Development and Disease

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.