DNA modification by methylation of the cytosine base is a fundamental epigenetic mechanism by which a range of developmental and cellular processes are regulated. It has been demonstrated that DNA methylation is essential for animal development, since inactivation of a constitutively expressed DNA methylation enzyme Dnmt1 in mice result in embryonic lethality. DNA methylation plays important roles in regulation of gene expression, suppression of viral infection, genomic imprinting and X chromosome inactivation. In recent years, numerous studies have also linked DNA methylation to various types of human cancer. However, the underlying mechanisms by which alterations in DNA methylation promotes cancer formation is large unknown. De novo methylation of unmodified DNA is a developmentally regulated process which is activated in early embryonic cells, but is inert in adult tissues. Aberrant activation of de novo methylation in adult tissues has been associated with transcriptional silencing of tumor suppressor genes in multiple tumors. Recently, a novel family of de novo DNA methyltransferase genes, termed Dnmt3a and Dnmt3b, have been cloned from both human and mouse. The mouse genes are expressed in embryonic stem cells, but weakly in adult somatic tissues. To investigate how Dnmt3 methyltransferases are regulated and what roles they play during mouse development, and to determine whether alterations in DNA methylation or Dnmt3 expression causes cancer, the following specific aims will be pursued.
Aim 1 : To generate Dnmt3-deficient mice using gene targeting methods and characterize their developmental defects.
Aim 2 : To analyze Dnmt3 expression during mouse development and identify cis-acting elements that regulate Dnmt3 transcription.
Aim 3 : To determine whether endogenous Dnmt3 expression is induced by oncogenic transformation, and the effect of Dnmt3 transgene expression on de novo methylation of CpG islands, oncogenic transformation, and tumor formation in nude mice.
Aim 4 : To evaluate the genetic effects of inactivation or ectopic expression of Dnmt3 in transgenic mice on tumor progression or suppression. The intestinal tumor model, the Min mice, will be used to test whether Dnmt3a and Dnmt3b have a role in intestinal polyp formation like Dnmt1. The molecular and genetic approaches described in this proposal will unravel the biological function of de novo methylation in development and cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA082389-03
Application #
6377347
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Program Officer
Okano, Paul
Project Start
1999-07-01
Project End
2004-04-30
Budget Start
2001-05-30
Budget End
2002-04-30
Support Year
3
Fiscal Year
2001
Total Cost
$223,583
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
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Dodge, Jonathan E; Okano, Masaki; Dick, Fred et al. (2005) Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem 280:17986-91
Sado, Takashi; Okano, Masaki; Li, En et al. (2004) De novo DNA methylation is dispensable for the initiation and propagation of X chromosome inactivation. Development 131:975-82
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Chen, Taiping; Ueda, Yoshihide; Dodge, Jonathan E et al. (2003) Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol Cell Biol 23:5594-605
Gribnau, Joost; Hochedlinger, Konrad; Hata, Ken et al. (2003) Asynchronous replication timing of imprinted loci is independent of DNA methylation, but consistent with differential subnuclear localization. Genes Dev 17:759-73
Lehnertz, Bernhard; Ueda, Yoshihide; Derijck, Alwin A H A et al. (2003) Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin. Curr Biol 13:1192-200
Chen, Taiping; Ueda, Yoshihide; Xie, Shaoping et al. (2002) A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J Biol Chem 277:38746-54

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