DND1 Mediates Epigenetic Reprogramming During Cell Cycle Arrest In Male Germ Cells

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ABSTRACT It is well known that prior to the establishment of adult spermatogonial stem cells (SSCs), the chromatin in male germ cells (MGCs) undergoes extensive epigenetic reprogramming during a long period of mitotic arrest (G0). However, a number of critical questions about this process remain unanswered. Do all MGCs undergo similar reprogramming or is the process selective for a specific sub-population that will give rise to the SSCs? How do changes in methylation, histone modifications, and chromatin structure silence developmental pathways and stabilize SSC fate? Do these changes depend on cell cycle arrest? Here, we propose to address these important questions through the lens of the RNA-binding protein (RBP) dead end 1 (DND1). DND1 is essential to maintain MGC identity since loss of Dnd1 function results in trans-differentiation of germ cells to somatic fates or disruption of G0 and formation of teratomas. We recently developed a transgenic mouse line in which a functional fusion protein between DND1 and GFP is expressed from the endogenous locus (Dnd1GFP). This reporter distinguishes two MGC populations during G0: DND1-lo cells and DND1-hi cells, which represent only 5-12% of the population. RNA-seq revealed that Dnd1 transcript levels as well as transcript levels for multiple epigenetic regulators are 5-10 fold higher in DND1-GFP-hi cells. Furthermore, RNA immunoprecipitation (RIP) using antibodies against DND1-GFP identified multiple epigenetic regulators that are targets of DND1 during G0. Among these targets are DNA methyltransferases (Dnmts), the enzyme Setdb1, that imposes the nuclear lamina associated repressive histone mark (H3K9me3), five Tudor domain proteins (Tdrds), and four actin dependent regulators (Smarcs) that function in association with a matrix scaffold. We hypothesize (1) that in DND1-hi cells, DND1 controls the post-transcriptional regulation of epigenetic modifiers by stabilizing their transcripts and gating translation and (2) that many of these epigenetic regulators function in association with the inner nuclear membrane (INM) dependent upon maintenance of GO. This project will test the significance of heterogeneity among MGCs, the critical function of the RBP, DND1 in epigenetic reprogramming, and the role of cell cycle arrest. In Aim 1, we will determine whether DND1-hi cells represent a clonal or transient population, and whether transcripts of epigenetic regulators are protected and differentially translated in DND1-hi or DND1-lo cells. In Aim 2, we will map epigenetic changes over time, and determine whether they differ in DND1-hi and DND1-lo cells. In Aim 3, we will determine whether chromatin domains undergoing change are localized to the nuclear lamins, and whether changes are disrupted by blocking association with lamins or disrupting cell cycle arrest. This project will address the significance of heterogeneity among MGCs and the role of cell cycle arrest and the nuclear lamins. We expect these results will uncover vulnerabilities to physiological, disease-related, or environmental conditions that alter epigenetic mechanisms and affect gametogenesis and male fertility.