Regulation of Male Germ Cell Development through DND1-Mediated Translation of Epigenetic Factors

项目总结

PROJECT SUMMARY Male germ cells (MGCs) are precursors to spermatogonial stem cells (SSCs), a stem cell population that both self-renews and differentiates, supplying spermatozoa for the entirety of a male’s reproductive lifespan. Disruptions in development of MGCs during fetal life leads to infertility in humans and mice. Just prior to birth, MGCs undergo a relatively long period of cellular quiescence. This G0 arrest phase is conserved between mice and humans and is considered an MGC reprogramming stage. During this time, MGCs lose their early GC fate and acquire the molecular profile of SSCs by undergoing vast epigenetic modifications. Although increases in mRNA levels of various epigenetic factors occur during G0, it is unclear how the transcripts of epigenetic factors are regulated post-transcriptionally throughout G0. DND1 is one of many RNA-binding proteins (RBPs) required for MGC development, and it is essential for both G0 arrest and for RNA expression of epigenetic regulators during G0. Transcript targets of DND1 during G0 include vital epigenetic regulators. Functionally, DND1 can promote transcript degradation, protect transcripts from degradation, or promote their translation. Because DND1 has multiple regulatory roles, it is unknown how DND1 post-transcriptionally regulates its target transcripts during G0 to ensure MGC-to-SSC development. In this proposal, how transcripts of epigenetic factors are post- transcriptionally regulated G0 in MGCs will be determined by focusing on the translational role of DND1, as supported by preliminary data. The central hypothesis is that an essential role of DND1 is to control when and where its target epigenetic regulators are translated during G0 to direct the reprogramming of MGCs into SSCs. To test this hypothesis, in Aim 1, DND1-bound transcripts that encode for epigenetic regulators and that are translated during G0 will be defined. The proteomes will be determined through LC/MS-TOF in two stages of G0 and will be cross-referenced to previously obtained DND1-target transcripts at their respective stages, creating DND1-directed translatomes. Epigenetic regulators in these translatomes will be assessed for nascent translation throughout G0 to determine their temporal translational regulation. In Aim 2, it will be determined where and how DND1 promotes the translation of its target transcripts. DND1-interacting proteins will be unbiasedly identified and using a variety of high-end microscopy tools, the localization of DND1, its target transcripts, and translational machinery will be determined. The insight provided by these aims will provide a novel paradigm for how an RBP regulates the epigenetic reprogramming of MGCS to ensure the development of SSCs. In addition to advancing scientific knowledge, this fellowship proposal also exhibits high training potential. The training plan describes a two-year blueprint designed to strengthen technical, professional, and conceptual skills, which will be implemented at Duke University in the laboratory of Dr. Blanche Capel. Overall, the enclosed research strategy and training plan can develop a trainee into an independent researcher while significantly advancing knowledge for improving reproductive health.