Protecting and Sustaining Germ Cell Identity

Project Summary/Abstract Germ cells have the extraordinary potential to generate every cell type in the body. Yet, the molecular program that protects and sustains this promise for totipotency in germ cells, which can last more than forty years in humans, is poorly understood. To face this challenge, germline regulators assume dual roles in maintaining germ cell identity: activating a germline transcriptional program while simultaneously protecting germ cells from reprogramming to a somatic cell fate. Indeed, genes involved in germline specification, such as the conserved RNA-binding protein Nanos and the transposable element (TE) regulator Piwi, are erroneously upregulated in certain human tumors. These tumors are thought to undergo soma-to-germline transformations to acquire a more immortal, germline-like state. However, a specific program for germ cell transcriptional activation has yet to be described, mainly because a ‘master-regulator transcription factor’ for germ cell fate is missing. This proposal uses a multipronged approach to systematically characterize the gene expression program in primordial germ cells (PGCs) and identify prime regulators of germ cell fate. The goal is to distinguish between a ‘default’ model, by which repression of the somatic program ‘allows’ the PGC program, and an ‘instructive’ model, by which PGC specification is actively controlled. In Aim1, we probe the instructive model to uncover the germline transcriptional program. By single-cell RNA sequencing, we have detected specific, temporally-regulated germline genes. To identify the transacting regulatory factors, we will use ATAC-sequencing, characterize known factors with matching binding activities, and identify potential new regulatory candidates for germline genes. In Aim2, we address the function of a critical regulator of germ cell fate, the conserved RNA regulator Nanos. Using the RNA target identification method hyper-TRIBE and ribosome profiling, we will identify Nanos targets and functionally distinguish between those targets that promote the germline program and/or repress the somatic program. Aim3 focuses on the piRNA pathway, which provides maternally inherited immunity against TE activity by controlling the transcription of TE elements, specifically in germ cells. Using a novel degradation strategy to specifically degrade components of the piRNA pathway in PGCs without impacting oogenesis, we will determine the role of this pathway for embryonic PGC gene expression beyond TE control. By integrating multiplex findings, this proposal will uncover the regulatory landscape of early germ cells, thereby providing a necessary understanding of the process of gonadogenesis and, ultimately, the survival of the species.