Mechanistic Roles of PUF-8 and Its Partners in Controlling Germline Stem Cell Maintenance and Differentiation in Caenorhabditis elegans

Abstract

Stem cells are highly regulated by conserved extrinsic and intrinsic regulators. Among these regulators, cell cycle regulators and PUF proteins play a crucial role in cell fate and the maintenance of stem cells; however, their functional interplay remains poorly understood. Of the 11 PUF proteins in C. elegans, PUF-8 is most closely related to human PUF proteins PUM1 and PUM2. Within the germline, PUF-8 collaborates with additional regulators to control germline stem cell (GSC) proliferation, differentiation, and maintenance. This research demonstrates that both CYB-3/CDK-1 and CYE-1/CDK-2, as well as PUF-8, are required for GSC maintenance during both early germline development and adult germline. RNAi-mediated knockdown of cyb-3, cdk-1, cye-1, or cdk-2 in puf-8 mutants results in proliferation and maintenance defects of GSCs. Additionally, PUF-8 and these cell cycle regulators protect meiotic cells from CED-4-mediated apoptosis. Furthermore, we elucidate the roles of PUF-8, CYB-3/CDK-1, and CYE-1/CDK-2 across three germline tumor models driven by ectopic proliferation, failure of meiotic entry, or dedifferentiation. In all three contexts, PUF-8 and these associated cell cycle regulators suppress germline tumor formation and maintenance. We further investigated dedifferentiation-mediated tumorigenesis and its regulation by the chemical tert-Butylhydroquinone (tBHQ) and R-loop repressors, RNH-1/2 and BRC-1/2. In puf-8 mutant germlines, dedifferentiation-mediated tumorigenesis depends on MPK-1 signaling. Interestingly, under high-glucose conditions, tBHQ increases the level of phosphorylated MPK-1b, thereby enhancing dedifferentiation-mediated tumorigenesis. The mechanisms by which glucose and tBHQ synergize to promote tumor formation remain unknown. We further show that R-loop repressors and PUF-8 are required to suppress aberrant R-loop accumulation, thereby preventing dedifferentiation-mediated tumorigenesis. Together, these results provide deeper insight into the interplay between dedifferentiation and tumorigenesis.