DESCRIPTION (provided by applicant): The goal of this research program is to define how an egg is produced, provisioned, and protected and how the egg is dramatically reorganized for the transition from oocyte to embryo. Analysis of this key window in development is accessible in Drosophila, and due to the conservation of regulators in development, studies in Drosophila will identify regulatory mechanisms and components that will shed light on fundamental features of early development, with implications for human health in understanding developmental defects and infertility. In addition, analysis of how a Drosophila egg is produced has provided models for the control of metazoan DNA replication, generating insights into how gene copy number can become altered in cancer cells. The proposed research will study the crucial processes of DNA replication, mRNA translation, and proteolysis, all of which are subjected to developmental control in the transition from oocyte to embryo. The regulation of replication origins has remained elusive in metazoans because it has not been possible to determine what defines an origin, to localize the essential Origin Recognition Complex (ORC), or to capture origin firing and its control. Drosophila offers the tremendous advantages of developmental control of origin activity and the ability to localize ORC in differentiated tissues to map origins The effect of differentiation and cell cycle modification on ORC localization will be determined, and the relationship between ORC binding, transcription and developmental repression of replication defined. Replication origins that bind ORC across a broad zone and undergo repeated rounds of origin firing in response to differentiation signals will be exploited to deciphr how replication initiation is controlled. The transition from the differentiated oocyte to the totipotent embryo occurs in the absence of transcription, involving rapid changes in osmolarity and extensive remodeling of the proteome by dramatic changes in mRNA translation and proteolysis. Regulators identified by genetic and proteomic screens indicate a crucial role for redox state and a glycine transporter in egg activation that will be delineated. The PNG kinase was shown to be a master regulator of mRNA translation specifically at this developmental transition, providing the link between completion of meiosis and control of mRNA translation in the egg. The mechanism(s) through which PNG activates translation will be explored by testing the hypotheses that it phosphorylates and inactivates translational repressors and affects the localization of mRNAs to RNP complexes in the egg. A unique form of the Anaphase Promoting Complex (APC), APCCort, specifically targets proteins for degradation whose removal is required for the change from meiosis to mitosis. The requirement for APCCort-targeted degradation of recently identified substrates that are critical in the oocyte-to-embryo transition will be evaluated, and the function of other components of the ubiquitin pathway expressed specifically in this developmental period will be defined.

PUBLIC HEALTH RELEVANCE: At the onset of development the totipotent embryo is formed as the differentiated oocyte is fertilized. The mechanisms leading to this profound change are being investigated, including how the protein content in the egg is reset by translation of mRNAs and regulated protein degradation, how changes in osmolality and redox trigger this transition, and how DNA replication is regulated. Proper regulation of the oocyte-to-embryo transition is essential for fertility and to prevent birth defects, and the understanding of regulation of DNA replication emerging from this work will provide insights into how gene copy number goes awry in cancer cells.