Comprehensive genome sequencing has the potential to dramatically improve our understanding of the genetic underpinnings of normal biology and disease states. Uncovering how complex genomes are epigenetically modified and identification of the protein and RNA mediators responsible is a critical next step towards understanding the dynamic changes that occur throughout development and differentiation. My interests lie in understanding this epigenetic regulation of mammalian genomes, and we use the earliest stages of mouse development as a model system. Although brief in time, preimplantation development is an extremely dynamic period during which major epigenetic remodeling occurs, and the very first cell fate decisions are made. These key biological processes require global, yet exquisitely precise chromatin remodeling. We utilize various RNAi approaches towards identification of genes that regulate these earliest epigenetic decisions that occur during oogenesis and preimplantation development.
Along with the genome sequences now available, extraordinary amounts of expression data are rapidly being produced. Several reports have defined large expression data sets during preimplantation development. We have used this information to assemble and validate lists of candidate genes with expression patterns that suggest important roles during preimplantation. Of particular interest are two expression patterns - genes which are expressed only during cleavage stages (A-P-A in figure), and those which are expressed in the egg and 1 cell zygote, but not thereafter (P-A-A). As these two expression patterns suggest roles during distinct developmental windows we use different approaches to assess the function of these genes.
Epigenetic regulation during preimplantation development begins with the remodeling of egg and sperm haploid genomes prior to pronuclear fusion. By the time of blastocyst formation (3.5 days post fertilization in the mouse), differential chromatin structure has been established at sites throughout the genome, including parent of origin imprinted domains as well as trophoblast and ICM specific gene expression. To capitalize on the manipulability of this early window of epigenetic reorganization, we knock-down candidate transcripts by injecting or electroporating long dsRNAs into single cell embryos, followed by in-vitro culture until the blastocyst stage.
Genome imprinting is an epigenetic mechanism resulting in differential transcriptional activity between the two parental alleles. It is well established that differential chromatin structure accompanies this parent of origin gene expression. More specifically, DNA hypo/hyper-methylation, and core histone modifications (acetylation/methylation) differ between the active and silent alleles. Disruption of any one of these chromatin modifications may result in loss of imprinting at particular loci, making imprinted genes sensitive “reporters” of epigenetic regulatory mechanisms. In addition to harboring the necessary polymorphisms for imprinting and X inactivation assays, the mouse strains used for the phenotypic screen will also carry an EGFP transgene driven by the Oct4 promoter, which is specifically expressed in the ICM at the blastocyst stage. We therefore screen for loss of imprinting, defects in trophoblast/ICM differentiation, as well as developmental arrest and morphological abnormalities all within the same embryos.
Another approach that our lab utilizes is the creation of mice that carry transgenes that knock-down genes of interest specifically in developing germ cells (egg and sperm). This approach allows us to explore the role of epigenetic regulatory genes prior to fertilization during germ cell development – a time when genome wide alterations are required to erase any and all somatic cell modifications, and prepare the diploid cell nucleus for the process of meiosis and haploid gamete production. This transgenic RNAi approach allows for allelic series of mice to be generated with each founding transgene eliciting various levels of gene specific knock down. Importantly, we have already generated phenotypes not only during oogenesis, but during preimplantation as well – indicating that this approach can identify gene functions which are truly epigenetic in origin –loss of function during oogenesis that result in defects several days later during preimplantation development.
We currently generate transgenic RNAi constructs that act during oocyte development, and are pursuing similar approaches in the male germ line to specifically knock down genes during spermatogenesis.