Regulation and functions of DNA polymerases in the Drosophila male germline stem cell lineage

About This Grant

In the process of asymmetric cell division (ACD), two daughter cells with identical genetic information, but distinct fates, result from one cell division. Adult stem cells can undergo ACD to produce one self-renewed stem cell and one differentiating daughter cell. This process allows for important physiological processes including tissue development, homeostasis, and healing. Regulation of proper stem cell division can be lost in aging as well as cancer and other chronic diseases. How stem cells regulate ACD is not fully understood, but has broad implications for the study of cell fate determination and cellular reprogramming. To study ACD in an in vivo adult stem cell lineage, the Chen lab uses the Drosophila male germline. Germline stem cells (GSCs) in the Drosophila testis divide to generate both a self-renewed GSC and a differentiating daughter cell, called a gonialblast, which divides symmetrically to produce spermatogonial cells (SGs) that eventually undergo meiosis and terminal differentiation into sperm. Our group previously showed that histones that existed in the cell before DNA replication are retained in the self-renewed GSC, while newly synthesized histones are enriched in the differentiating daughter cell. The lab then discovered that during S- phase in GSCs, old histones are biased to the leading strand while new histones are biased to the lagging strand. Interesting, the catalytic subunits of both lagging strand enriched polymerase complexes, Polα and Polδ, are present at a lower level in GSCs as compared to SGs, while the leading strand enriched polymerase, Polε, shows comparable levels. Compromising Polα either pharmacologically (by an inhibitor) or genetically (polα+- flies), induces asymmetry in histone incorporation in SGs. However, how this differential expression of Polα and Polδ, but not Polε, is regulated is unknown. According to my preliminary data, polα and polε RNA levels reflect the protein level trend, and a stepwise increase in polα RNA across SG divisions was noted. I hypothesize that GSC transcriptionally repress polα, and this repression is lost as SGs divide. I will determine what cis-regulatory elements are responsible for this differential expression using a reporter assay. I will integrate transcription factor motif analysis, genomic data, and transcriptomic data to identify candidate trans-acting factors, which I can test further using molecular and genetic assays. In my second aim, I will investigate the functional outcome of reduced Polδ levels. The lab found that both polδ+/- and polα+/- flies display sustained fertility during aging. polα+/- flies also demonstrate enhanced regeneration in the germline. I will characterize the cell biology and morphology of polδ+/- testes during aging and test their regenerative potential using a genetic ablation experiment. The Chen lab has the expertise and resources necessary for me to carry out this proposed work. Between Dr. Chen’s mentorship and both academic and career support from the Johns Hopkins Department of Biology, I am confident I will have a successful doctoral research career and my findings will contribute to the NIH’s public health goals as well as benefit the broader scientific community.