Imprinted genes are a subset of genes that are uniquely expressed from only one parental allele. Monoallelic expression of these genes is particularly important for mammalian development. Notably, inappropriate expression of specific imprinted genes leads to severe developmental disorders in humans, e.g. Beckwith-Wiedemann syndrome. Allele-specific DNA methylation is erased and reestablished during gametogenesis at regulatory elements, known as imprinting control regions (ICRs). DNA methylation at ICRs controls imprinted gene expression in the resultant embryo. The main objective of this proposal is to investigate how DNA demethylation occurs and allows for the proper acquisition of sex-specific DNA methylation patterns that regulate imprinted gene expression. While recent studies from the Bartolomei lab and others implicate the enzyme, TET1, in the DNA demethylation process, it is not known if TET1 plays a role in the tight control of imprinted gene expression.
Aim 1 will test the hypothesis that TET1 is required for the proper erasure of DNA methylation at ICRs and monoallelic imprinted gene expression. First, the methylation status of ICRs in primordial and mature germ cells will be assessed using a Tet1-/- mouse model. To further elucidate the developmental consequences of the loss of Tet1 function, allele-specific expression of imprinted genes will be analyzed in somatic tissues of maternally- or paternally-inherited Tet1-/- F1 hybrid offspring.
Aim 2 will test the hypothesis that repressive post-translational histone modifications prevent the binding of TET1. Chromatin immunoprecipitation will be used to determine if TET1 binding in primordial germ cells is inversely correlated with H3K9me2/3. To directly test if H3K9me2/3 prevents TET1 binding and function, the normal epigenetic state at ICRs will be experimentally perturbed using CRISPR/dCAS9 fusion proteins in an in vitro model of primordial germ cells. In total, this proposal will determine the molecular mechanisms that underlie the function of TET1 in the epigenetic reprogramming of both the male and female germlines and may lead to insights into the etiology of human imprinting disorders.
The proposed work in this application aims to understand how the protein, TET1, affects the unique expression of certain genes, called imprinted genes, which are crucial for normal mammalian development and growth. Additionally, this work seeks to understand molecular mechanisms that underlie TET1 recruitment to its relevant binding sites at the DNA regions that control imprinted gene expression during sperm/oocyte development. Abnormalities of the TET1 protein or the processes that allow TET1 to find its DNA binding sites may lead to novel clues relating to causes of human imprinting syndromes.
| SanMiguel, Jennifer M; Bartolomei, Marisa S (2018) DNA methylation dynamics of genomic imprinting in mouse development. Biol Reprod 99:252-262 |
| SanMiguel, Jennifer M; Abramowitz, Lara K; Bartolomei, Marisa S (2018) Imprinted gene dysregulation in a Tet1 null mouse model is stochastic and variable in the germline and offspring. Development 145: |
