The mechanisms governing the transition from a terminally differentiated oocyte to a totipotent zygote remain poorly understood. During the previous funding period, we cloned and characterized a highly-abundant oocyte and embryo-restricted gene, peptidylarginine deiminase 6 (PADI6), from the murine egg proteome and found that it represents a novel member of an enzyme family (PADI) that converts protein arginine residues to citrulline. Our ongoing analysis of the PADI6 -/- phenotype indicates that PADI6 -/- females are infertile due to an arrest in embryogenesis at the two-cell stage, thus demonstrating that PADI6 represents a novel maternal effect gene. At the ultrastructural level, PADI6 localizes to an egg and embryo-restricted structure, the cytoskeletal sheets (CSS), that occupies over 15% of the cytoplasm in many mammals. While the function of this cytokeratin-containing structure is unknown, the CSS form during oocyte growth and undergo dramatic reorganizations at critical developmental time points;suggestive of a role in embryonic reprogramming. Strikingly, preliminary ultrastructural analysis reveals a complete dispersal of the cytoskeletal sheets in PADI6 -/- oocytes and eggs while most other structures appear normal. We also found that transcriptional activity in PADI6 -/- two-cell embryos is severely compromised, suggesting that the PADI6 defect arises due to failure to activate embryonic transcription. Based on our preliminary findings, we hypothesize that, in the oocyte, PADI6 interacts with cytokeratin via its N-terminal domain leading to nucleation of the dispersed CSS components. Following fertilization, we hypothesize that PADI6 becomes activated (most likely by calcium signaling) and citrullinates cytokeratin via its C-terminus, leading to CSS reorganization and a stage-dependent release of associated proteins which are required for embryonic genome activation (EGA).
The specific aims of this application are to: 1) Test the hypothesis that PADI6 is required for nucleation of CSS components into the mature 60 nm complex in the oocyte. 2) Test the hypothesis that PADI6 plays a role in cytoplasmic-to-nuclear signaling events in the early embryo prior to genome activation. 3) Test the hypothesis that citrullination of cytoskeletal sheet proteins by PADI6 is required for CSS anastomoses and for early development.
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