Deciphering Mechanisms of Early Meiotic Gene Expression Through Ume6 and Ime1

The process of gametogenesis is orchestrated by a dynamic gene expression program, where a vital subset constitutes the early meiotic genes (EMGs). In budding yeast, the transcription factor Ume6 represses EMG expression during mitotic growth. However, during the transition from mitotic to meiotic cell fate, EMGs are activated in response to the transcriptional regulator Ime1 through its interaction with Ume6. While it is known that binding of Ime1 to Ume6 promotes EMG expression, the mechanism of EMG activation remains elusive. Two competing models have been proposed whereby Ime1 either forms an activator complex with Ume6 or promotes Ume6 degradation. Here, we resolve this controversy using a combination of depletion and tethering strategies to functionally characterize both Ume6 and Ime1 (Chapter 2 and Chapter 3)./nMuch of the research surrounding Ume6 function, and the genes it regulates, has come from using a null allele (ume6∆). Accordingly, constitutive loss of UME6 function leads to derepression of several meiosis specific genes during mitosis. Expression of these meiotic genes during the mitotic cell cycle causes conflicts in cellular machinery and leads to pleiotropic consequences. Instead of ume6∆ to assess UME6 function, here we leverage the auxin inducible degron (AID) system to construct a depletable allele of UME6 (Ume6-AID). This allele of UME6 maintains repression of its targets during the mitotic cell cycle. Using this approach, we identify the set of genes that are directly regulated by Ume6, including UME6 itself (Chapter 2)./nWith the development of Ume6-AID, we next investigated the functional relationship between Ume6 and Ime1 by combining our Ume6-AID with a method of synchronizing expression for IME1, which encodes the meiotic transcription factor, and IME4, which encodes an mRNA N6-adenosine methyltransferase (Chapter 3). This increased control allowed careful monitoring of how Ume6 responds to IME1 and IME4 expression and allowed functional dissection of Ume6’s role in the mechanism of early meiotic gene (EMG) expression. We find that, while Ume6 protein levels increase in response to Ime1, Ume6 degradation occurs much later in meiosis, in a manner dependent on another meiotic transcription factor called Ndt80. Importantly, we found that depletion of Ume6 shortly before meiotic entry is detrimental to EMG activation and gamete formation. Finally, to explore the functional role of Ime1 in EMG expression, we employed a tethering strategy. We find that tethering of Ume6 to a heterologous activation domain is sufficient to trigger EMG expression and produce viable gametes in the absence of Ime1. These data indicate that Ime1 and Ume6 form an activator complex in meiosis. While Ume6 is indispensable for EMG expression, Ime1 primarily serves as a transactivator for Ume6./nThis dissertation unifies observations from two disparate models involving Ime1 and Ume6 and their involvement in meiotic initiation. Our work unveils the impact Ime1 has on Ume6 through its binding to Ume6 and how this influences EMG expression. In doing so we elevate Ume6 to a primary determinant of cell state through its exchange of transcriptional cofactors and significantly advance our understanding of meiotic gene regulation.

Advisor: Ünal, Elçin