Our goal is to understand epigenetic regulation of gene expression in mammalian development and genetic disease. We focus on the role of DNA methylation in cancer, genomic imprinting, X chromosome inactivation, and the function of methylation in the postnatal brain. We use gene targeting methods to generate embryonic stem cells and mice with targeted gene deletions. Much of our earlier work has been based upon the targeted inactivation of the Dnmt1 gene, which encodes the only known mammalian DNA methyltransferase (MTase). Because DNA methylation deficiency causes early embryonic lethality, we have generated lineage specific knockouts of the gene to study its role in late events such as cancer and the postnatal brain. Finally, we are using mammalian cloning approaches to distinguish epigenetic and genetic alterations of the genome, which may be part of normal or abnormal development.
DNA methylation The targeted inactivation of the Dnmt gene results in embryonic lethality and thus established the essential role for DNA methylation in mammals. The Cre-mediated inducible Dnmt1 knockout in fibroblasts induces rapid apoptosis within one round of cell division even before any detectable demethylation. This suggests a direct link between DNA methylation and programmed cell death. Lineage specific deletion of Dnmt in different organs is being used to assess the role of methylation in normal development and cancer (see below).
Genomic imprinting We have shown that monoallelic expression of imprinted genes depends on DNA methylation. It has been proposed that imprinting functions specifically in early mammalian development, or alternatively, has no intrinsic role in development but is the result of a "tug of war" between the maternal and paternal genomes. To test whether imprinting has an intrinsic role in development, we are generating "non-imprinted" mice using two approaches. First, the Dnmt gene will be inactivated and reactivated during gametogenesis and embryogenesis using controlled gene deletion and activation to remove methylation imprints from the genome without causing lethality. Second, mice are being cloned from "non-imprinted" ES cells by nuclear transplantation.
X-inactivation The cloning of mammals requires the reprogramming of the genome from an epigenetic state characteristic for the somatic donor cell to one which is appropriate for directing early embryonic development. Most clones die after birth and we are interested in assessing whether poor survival of clones may be caused by faulty reprogramming. X-inactivation is a complex process where one of the two X chromosomes of a female cell is inactivated. We tested whether this epigenetic silencing of the X can be reversed by nuclear cloning and found that transfer of a female somatic nucleus into an oocyte fully reactivates the inactive X. Thus, faulty reactivation of the X chromosome does not impede survival of clones. We are now investigating whether the expression of other genes, in particular that of imprinted genes, is abnormal in cloned embryos.
Cancer The involvement of DNA methylation in cancer has been controversial: both hypomethylation as well as hypermethylation have been associated with malignant transformation. Genomic hypomethylation is a widely observed early step in human tumorigenesis. Animals carrying a mutation of Dnmt1 are hypomethylated and show a substantial increase in mutation rate. The mutations are caused by enhanced mitotic recombination and evoke aggressive leukemia induction in most animals, suggesting that hypomethylation may provide the tumor cell with a mechanism to efficiently delete tumor suppressor genes by LOH. Dnmt1 deletion in fibroblasts results in extensive demethylation. We have used gene chip analysis for genome wide expression profiling and found that about 10% of all genes become significantly de-repressed. This is clear evidence for a role of methylation in developmental gene silencing. We are now generating mice with conditional mutations in the Dnmt3 genes to study the role of de novo methylation on development and cancer.
DNA methylation and brain function MTase is highly expressed in the brain, particularly in hippocampus and cerebellum. To study the potential role of the enzyme in postnatal brain function, we are deriving mice carrying a Dnmt1 deletion in specific regions of the brain. Of particular interest is a mutation of MeCP2, a major methyl-DNA binding protein that acts as a transcriptional repressor. Mutations in this gene have recently been identified in humans with Rett syndrome, a frequent cause of severe mental retardation in women. Interestingly, Rett girls are normal up to one year of age and then rapidly regress (loss of speech and voluntary movement, autism, severe mental retardation). We have generated MeCP2 conditional mutations in mice and found that mutant animals recapitulate the human phenotype. Our goal is to use these animals to understand the pathology of the disease and to design therapeutic strategies which would prevent the phenotype from developing.
Neurotrophins and the CNS Finally, we are investigating the function in the CNS of neurotrophins such as BDNF and NT-3, which are highly expressed in specific regions of the brain. However, deletion of these genes does not result in a CNS defect. To study the role of neurotrophins in the postnatal brain, we have generated lineage specific mutations of BDNF and NT-3.