Conserved regulation of the switch from proliferation to differentiation in the germ line stem cell lineage The switch from proliferation to differentiation is a key regulatory point in adult stem cell lineages, crucial for tissue maintenance and repair. Failure of this switch may contribute to genesis of cancer. Here we study the molecular mechanisms underlying a developmentally programmed transition from proliferation to differentiation in the germ cell lineage, where the switch from mitosis to meiosis is a defining event. Drosophila bgcn encodes an RNA binding protein required cell autonomously for spermatogonia to stop proliferation and enter meiosis. We created mice null mutant for the mouse homolog of bgcn and discovered that genetic mechanisms that underlie a clean switch from mitosis to meiosis are deeply conserved from Drosophila to mammals. Male germ cells in Bgcn mutant mice execute the spermatogonial mitotic divisions and initiate meiosis, turning on Stra8 and undergoing premeiotic DNA replication, but fail to stably implement the program for meiotic prophase. Instead the nascent spermatocytes show only low or transient expression of number of meiotic markers, fail to properly turn off several mitotic transcripts, prematurely enter an ectopic mitosis, then die. We propose to capitalize on these discoveries to map the regulatory circuitry controlling the switch from mitosis to meiosis in Drosophila males and explore how a homologous RNA binding protein, acting with different partners, enforces a clean switch from the mitotic program to the meiotic program in mammals. Using the short life span and powerful genetic tools in Drosophila, we identified the main target of repression by Bgcn and its co-factor Bam for the switch to meiosis as the RNA binding protein HOW, homolog of mammalian Quaking. We will investigate if Drosophila Bgcn and Bam directly regulate HOW mRNA translation or stability, mapping the HOW mRNA sequences required for these regulators to bind. To discover how HOW maintains the spermatogonial state, we will test the hypothesis that HOW prevents expression of a TGFB-class ligand required to signal to somatic cells that the germ cells are ready to progress. In parallel, in an unbiased approach to identify candidate substrates of HOW we will analyze RNA-Seq data to identify transcripts alternatively spliced in spermatogonia vs spermatocytes that have conserved HOW binding sites, test whether they co-immunoprecipitate with HOW from spermatogonia, then assess function in spermatogonia by genetic assays in Drosophila. To discover how mammalian Bgcn ensures an effective switch from mitosis to meiosis, we will investigate whether mBgcn targets mitotic transcripts that we discovered it binds for destruction or translational repression in young spermatocytes. To investigate how Bgcn, with its partner Meioc, may act indirectly to allow stable accumulation of meiotic transcripts, we will test a model based on our discovery that mBgcn binds certain spermatocyte specific piRNA precursors and is required for their accumulation. Our results will reveal molecular mechanisms that underlie the surprising discovery that RNA binding proteins play key roles in ensuring a clean switch from proliferation to differentiation in the male germ line stem cell lineage.
statement: The results of the proposed studies will reveal how a deeply conserved mechanism based on posttranscriptional control regulates the fundamental switch from proliferation to differentiation in the male germ line adult stem cell lineage. The mechanistic knowledge we obtain will provide new understanding of how mRNA binding proteins act and interact to control the fundamental switch from mitosis to meiosis essential for sexual reproduction and reveal the role of a component of the ?epitranscriptome? of post-transcriptional RNA modifications and a cell-type and stage-specific ?reader? of the modification in regulating a key developmentally programmed cell state change.
