Mechanisms of demethylation in primordial germ cells and the importance of stage-specific demethylation in safeguarding against precocious differentiation
Primordial germ cells (PGCs) are the cellular precursors for mature gametes which are responsible for giving rise to embryonic development and the next generation of PGCs. During development, proper PGC differentiation results in high quality gametes, which are essential for normal development and future child health. Problems during PGC differentiation can lead to impaired fertility, poor quality germ cells, or developmental defects in the next generation. One of the essential events that occurs during PGC development is whole-genome reprogramming of DNA methylation. The reprogramming of DNA methylation in the context of PGC development is required for appropriate cell lineage differentiation. This process is essential in establishing the correct epigenetic landscape which will impact differentiation, and maturation of PGCs. My goal is to focus on two aspects of genome-wide reprogramming in Primordial Germ Cells (PGCs). First, the molecular mechanisms of DNA demethylation during the gonadal stage of development, as well as the mechanisms involved protecting specific loci from demethylation in order to allow for correct temporal expression of germ cell genes
Primordial germ cells (PGCs) undergo genome-wide demethylation in two distinct stages. Stage 1 consists of global demethylation before embryonic (e) day e9.5 of mouse development. Stage 2 The second phase occurs once PGCs colonize the genital ridge between e10.5-e13.5, and happens in a temporal and locus-specific manner. Results indicate that the second phase is regulated in part by Ten eleven translocation (Tet) protein Tet 1, and conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) at specific loci. The major working model for Tet-dependent DNA demethylation involves replication-coupled loss of methylated cytosines from the genome. However an alternate model would predict active removal of 5hmC at specific loci independent of cell division.
In order to address this directly, we have established a new organ culture model involving the growth of dissected aorta/gonad/mesonephros (AGM) tissues isolated from the mouse embryo at e10.5. During three days of organ culture, we show that PGCs divide on average three times. We also show that in the background of global hypomethylation established in phase 1, PGCs isolated from the organ culture undergo locus-specific DNA demethylation, and 5hmC reorganization, and this occurs within three days. Using this model we have targeted the PGC cell cycle using a P-AKT inhibitor, and have determined that imprint erasure can happen in proliferation dependent and independent ways depending on the genomic locus
Alternatively, during the removal of DNA methylation in stage 1, some loci are protected from demethylation and the mechanism for this process remains unknown. In the current study we tested the hypothesis that Dnmt1 is responsible for maintaining methylation by being recruited at specific genomic sites during whole genome demethylation. To address this, we created a conditional germline knockout of Dnmt1. Analysis of Dnmt1 conditional knockout (DCKO) PGCs revealed that Dnmt1 is the major methyltransferase that functions during whole genome demethylation to maintain DNA methylation at discreet genomic regions including intracisternal A particle (IAP) transposons, as well as maternal and paternal imprinting control centers. Furthermore, the absence of Dnmt1 results in precocious differentiation that leads to germ cell loss in both male and female embryos. Taken together, we propose a model in which maintenance of cytosine methylation by Dnmt1 is essential to maintain cytosine methylation at discreet regions of the genome during whole genome DNA methylation reprogramming.