DNA methylation is an epigenetic modification involved in transcriptional regulation of genes involved in
development and differentiation, and its deregulation contributes to human pathogenesis. It is catalyzed by the
family of DNA methyltransferases including catalytically active Dnmt1, Dnmt3a, Dnmt3b. DNA methylation
plays a major role in preimplantation development in mice. To establish a new epigenome, the mouse zygotic
genome undergoes epigenetic reprogramming, including global DNA demethylation at the 8-cell stage. Upon
implantation, a wave of de novo methylation in epiblast cells mediated by de novo enzymes Dnmt3a and
Dnmt3b results in new methylation patterns maintained by Dnmt1 that form a basis for tissue-specific
expression and differentiation.
Dnmt3b regulates developmental and imprinted genes, X chromosome inactivation, pericentromeric regions,
gene bodies and other genomic regions. Its importance in mouse development was demonstrated by
embryonic lethality of Dnmt3b-/- mice. We recently found that Dnmt3bCI/CI mice expressing catalytically inactive
Dnmt3bCI protein survived both pre- and postnatal development. Molecular analysis suggested that accessory
function - the ability to recruit other Dnmts to proper genomic loci – of Dnmt3b rather than its catalytic activity,
is important for methylation and survival. Here we hypothesize that Dnmt3b is a multifaceted protein whose
various activities involved in methylation affect pre- and postnatal development and are critical to prevent
disease formation in mice. To test this hypothesis, in Aim 1 we analyze global methylation and expression at
different stages of development in mice lacking various Dnmt activities to determine the scope of Dnmt3b’s
accessory function in Dnmt3a-mediated de novo methylation in vivo as well as regulation of transcription of
various genomic features including gene bodies, germline genes and transposons. In Aim 2, will test the ability
of Dnmt3b to complex with other Dnmts and contribute to de novo methylation induced by other Dnmts in
Dnmt1-/-;Dnmt3a-/-;Dnmt3b-/- triple knockout mouse embryonic stem cells. In addition, we will genetically test
the importance of Dnmt3a and Dnmt3l for Dnmt3b’s accessory function and validate our data in a human cell
line. In Aim 3, we will perform longitudinal study of Dnmt3b+/+ and Dnmt3bCI/CI mice conceived through the use
of in vitro fertilization (IVF) technique to analyze disease development, Dnmt levels, the rate of methylation and
gene expression errors, as well as their persistence over time.
Collectively, our studies will reveal physiological relevance of Dnmt3b activities in mouse development,
uncover basic mechanisms utilizing Dnmt3b functions and their involvement in IVF. Our results could result in
changes in Assisted Reproductive Technologies (ART) and affect the focus of preventive care for ART-
conceived individuals.