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PRMT5 is an essential survival factor for ground state pluripotency and primordial germ cells: From primordial germ cell differentiation in vitro to mammalian germline in vivo

  • Author(s): Li, Ziwei
  • Advisor(s): Clark, Amander T
  • et al.
Abstract

Our ancestors and our children are linked by a single, special cell lineage called the germline. Germ cells are tasked with the role to accurately pass DNA from one generation to the next. In today's society infertility is an important health concern as it is estimated to affect 10% of the reproductive age population. In many cases infertility can be traced to abnormal germline cell development. The early events of germline formation are difficult to study because of limited materials, especially low germ cell numbers in the process of embryogenesis.

To overcome the challenge of limited materials, an in vitro method to derive cell types faithfully reporting in vivo phenotypes needs to be devised to facilitate the research process. The key results from the in vitro model should then be evaluated carefully in vivo to show relevance in development. My thesis first focused on establishing an in vitro model to recapitulate early development of germline, by differentiating embryonic stem cells (ESCs) to form in vitro primordial germ cells (iPGCs), in a dish. This method allows good number of germ cells to be produced for molecular and biochemical studies. Using the in vitro model, key germline modifiers for ESC maintenance and germ cell formation were identified, one of which is protein arginine methyltransferase 5 (PRMT5).

PRMT5 is a type II arginine methyltransferase (PRMT) that modifies symmetrical dimethylated arginines (SDMAs) on proteins, which substrates include histone H2A and H4, as well as the Sm proteins involved in RNA splicing. However, the molecular function of PRMT5 in ground state ESCs remains unknown. In our study, for the first time, we generated an inducible knock out of Prmt5 in ESCs cultured in ground state pluripotency (cultured with 2i inhibitors and leukemia inhibitory factor). We characterized PRMT5's role in ESCs through the method of paired-end RNA sequencing to show that PRMT5 affects cell survival, proliferation and chromatin organization etc., by affecting RNA spicing. By generating a conditional knock out to specifically deplete PRMT5 in the germline in vivo, we showed that PRMT5 is necessary for mammalian PGC formation.

Taken together, we developed a differentiation system to ask questions about key factors regulating ESC and germline development. One of the key factors we identified is PRMT5. The role of PRMT5 was scrutinized using paired-end RNA sequencing in the inducible knock out ESCs, suggesting that PRMT5 is necessary to ensure normal splicing in "RNA processing", "DNA damage response" and "chromatin modification" for cell to survive. Finally, we utilized in vivo mouse model to validate events that happen in vitro, showing that PRMT5 is indeed a survival factor both for ground state pluripotency and mammalian germline.

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