Cryo-electron microscopy study of yeast RENT and SIR complexes

Abstract

In budding yeast, Cdc14 is a master phosphatase that orchestrates various events essential for mitotic exit. Through targeting a broad range of substrates, including Sic1, ORC, Cdc6, and Mcm2-7, Cdc14 counteracts CDK activities and resets the competency of replication licensing during mitotic exit. Cdc14 is sequestered by its inhibitor Net1 in the nucleolus for most of the cell cycle and is only released upon anaphase onset and activated sequentially through mitotic exit network pathways. In the nucleolus, Net1 associates with Cdc14 and Sir2, a NAD+ dependent histone deacetylase, to form a complex named RENT (regulator of nucleolar silencing and telophase). This complex is further recruited onto ribosomal DNA by Fob1 and RNA polymerase I, which in turn mediates rDNA silencing. However, the detailed mechanisms of Cdc14 sequestration by Net1 and the function of RENT complex in regulating gene silencing are not well understood at a molecular level. The major reason can be attributed to the scant high-resolution structural information available about RENT complex. In this study, I aim to reconstitute the RENT in vitro for structural determination using cryo electron microscopy (cryo-EM). I have successfully purified all RENT components, Net1, Cdc14, and Sir2 from either E.coli or yeast expression system, and optimized the conditions for complex assembly. Our preliminary EM analysis indicated that the assembled RENT complex is quite heterogenous and very dynamic. To improve the homogeneity and stability of the complex, I have made various truncation mutants of the RENT components with their potential disordered regions removed. In addition, I found that Fob1, an important factor in regulating replication-transcription collision, shows a direct interaction with Net1 and Sir2, likely contributing to stabilize the RENT complex on DNA or chromatin. The RENT-Fob1 complex will be assembled on rDNA template for cryo-EM analysis to provide structural insights into the function of RENT in mediating rDNA silencing.

Replication licensing and gene silencing are coupled at mating type loci where origin recognition complex (ORC) mediates the recruitment of silent information regulator (SIR) complex to mediate the formation of silent heterochromatin. To better understand how SIR complexes is recruited onto the mating type loci to function, I sought to reconstitute silent chromatin using purified proteins. So far, I have purified Sir2-Sir4 heterodimer and Sir3, and obtained in vitro assembled nucleosome. Furthermore, I found that both Sir2-Sir4 and Sir3 can be successfully assembled onto the nucleosome in combination or individually. Next, I will try to optimize the protocol for large-scale assembly reaction to obtain enough amount of the SIR-nucleosome complexes for structural determination using cryo-EM. The information derived from this study is expected to provide crucial structural information about the function of the SIR complex in mediating silencing chromatin.