Widespread Association of the Argonaute Protein AGO2 with Meiotic Chromatin suggests a Distinct Nuclear Function in Mammalian Male Reproduction
Date of Award
Doctor of Philosophy (PhD)
A variety of mechanisms in different compartments of the cell cooperate to regulate gene expression. Some mechanisms are specific to certain compartments, such as the regulation of transcription by changes in histone modifications in the nucleus or protein translation repression in the cytoplasm. Other mechanisms, such as gene regulation by Argonaute proteins, span multiple compartments. Argonaute 2 (AGO2) is the best characterized member of the Argonaute family. It is a ubiquitously expressed, key protein in the cytoplasmic regulation of RNA transcripts, vital to the development and homeostasis of organisms. Recent studies in lower organisms have implicated AGO2 function in the nucleus in regulation of gene expression at the transcriptional and post-transcriptional stages; however, this function remains poorly characterized in mammals. In this dissertation, I explore the nuclear role of AGO2 using mouse male germ cells as a model. I demonstrate that nuclear AGO2 binds RNAs in a developmental stage-specific manner and participates in unique protein interactions separate from its interactions in the cytoplasm of spermatogenic cells. Consistent with studies in other cell types, I also found that AGO2 interacts with the genome during spermatogenesis particularly at introns and intergenic regions. Conditional knockout (cKO) of Ago2 caused reduced sperm count, abnormal sperm head morphology, and a decrease in pup survival at weaning. Further analysis of Ago2 cKO germ cells found transcriptional changes and protein downregulation, opposite to the inhibitory role of AGO2 in the cytoplasm. Comparison of the downregulated proteins, transcripts bound by nuclear AGO2, and interactions with genomic loci highlighted a subset of genes where AGO2 may be regulating gene expression through multiple mechanisms. We focused on several genes in this subset that are implicated in male fertility and sperm head morphology to link the molecular changes we found with the phenotypic changes observed in Ago2 cKO mice. Together, our data supports a unique role for nuclear AGO2 in the regulation of gene expression during spermatogenesis through its interaction with transcripts and chromatin in a developmental stage specific manner. Our data diverges from previous work in the field in finding phenotypic changes in our Ago2 cKO male mice: downregulation at the transcript and protein level of developmentally important genes for spermatogenesis, sperm with malformed sperm heads, and death of offspring. These results demonstrate the importance of AGO2 in spermatogenesis and developing offspring and provide a model that can be used in future studies. Additionally, the datasets in this study provide a resource for the field to further our understanding of gene regulation by AGO2 in mammals.
Griffin, Kimberly Nicole, "Widespread Association of the Argonaute Protein AGO2 with Meiotic Chromatin suggests a Distinct Nuclear Function in Mammalian Male Reproduction" (2022). Yale Graduate School of Arts and Sciences Dissertations. 598.