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

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.

Grant Number: 5R01HD103064-05
NIH Institute/Center: NIH
Principal Investigator: Blanche Capel