Identification of Novel Regulators of Entry into the Meiotic Program

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How do somatic, mitotically dividing early germ cells enter meiosis and become haploid germ cells? How do cells switch from mitotic division to meiotic division, and how are meiotic cell divisions prevented in mitotically dividing cells? Only very few factors that control the fundamental switch from mitosis to meiosis are known, despite the obvious importance of this switch for all higher eukaryotic life. The generation of gametes and functional reproductive tissue depends on this switch. The lack of knowledge in this area is in part due to difficulty in identifying cells that have just entered the meiotic program. This pilot project aims at identifying hitherto unknown factors that prevent irregular switching from mitotic into meiotic cell divisions. The removal of this factor will be the driving force that allows cells to enter meiosis in a timely manner during development and self-renewal. We have performed an esiRNA high throughput screen (HTS) using our recently generated embryonic stem (ES) cell reporter line, which specifically indicates the onset of the meiotic program by GFP expression. Eliminating factors that prevent entry into meiosis turn these cells GFP-positive, allowing us to identify such negative regulators of entry into the meiotic program. After performing a high-throughput primary screen and a secondary validation screen using an esiRNA library representing >13.000 genes, we have identified 279 candidate genes. In the one year pilot project requested to be funded, these genes will be investigated by a variety of methods including bioinformatics, knowledge-based assessment, RT-PCR expression studies in pre-meiotic and meiotic cells, and the most promising hits will be further analyzed by using or raising antibodies for protein expression, protein interaction and protein localization studies. In the mid- and long-term, after successful completion of the pilot project, we aim at fully characterizing the most promising top hits molecularly and in mouse models and expect to thereby contribute significant new insights into our understanding of the mitosis-to-meiosis transition. These discoveries will allow a deeper understanding of germ cell disease and abnormalities in the murine model, and may also have further impact in the clinical setting by future extension of the analysis to human patients.