Mechanism and Regulation of Replication-Coupled Asymmetric Histone Incorporation in Drosophila Male Germline Stem Cells

PROJECT SUMMARY Adult stem cells often divide asymmetrically to form one self-renewing stem cell and a differentiating daughter cell. This process is important to maintain homeostasis and produce cells with the correct fate. Because both daughter cells inherit the same genetic information, it is logically inferred that epigenetics play a key role in the differing cell fate decision. How epigenetic information is parsed in such an asymmetric way, however, is a significant question. One key epigenetic component are histones, and the Chen Lab previously discovered that histones are inherited asymmetrically with respect to preexisting (old) and newly synthesized (new) histones during the division of Drosophila male germline stem cells (GSCs), the first division of spermatogenesis. Old histones are preferentially inherited by the self-renewing GSC while new histones go to the differentiating cell. This suggests a potential mechanism by which asymmetric epigenetic information could be inherited in such a way to determine different cell fates in genetically identical daughter cells. Further, previous work in the lab has found that the histone asymmetry is established during S-phase by asymmetric histone partitioning at the DNA replication fork. This suggests S phase and DNA replication play a potentially large role in determining cell fate, but the precise coordination of factors determining when and where asymmetry occurs remains elusive. It is of interest to study how this asymmetry is established, which genomic regions are subject to asymmetry, and how this process can be regulated. Here I propose studying the role of two important and interconnected aspects impacting DNA replication: chromatin environment and replication timing. I have preliminary evidence that histone modifications associated with different types of chromatin (eg. euchromatin vs. facultative heterochromatin) incorporate at varying degrees of asymmetry in GSCs, suggesting histone recycling patterns are chromatin context-dependent. I propose systematically studying different histone modifications in GSCs and non-stem cells using single-molecule chromatin fibers combined with super resolution microscopy that can distinguish individual strands at the replication fork. Further, because timing during S phase is inherently linked to what type of chromatin is being replicated, I propose investigating the relationship between histone asymmetry and the DNA replication timing program. First, I plan to treat Drosophila testes with hydroxyurea, which will synchronize cells at the start of S phase. Measuring histone incorporation at different times following release into S phase will determine how different stages of S phase may experience different degrees of asymmetry. I also propose studying total histone asymmetry in fly lines lacking the master timing regulator Rif1 to determine if the DNA replication timing program is necessary for histone asymmetries to be established. The results of this project will contribute to a model of how histone asymmetry is achieved and what parts of the genome are most responsible for differential cell fate decisions following asymmetric epigenetic inheritance, paving the way for a better overall understanding of developmental biology, stem cell biology, and cell fate specification. This knowledge will be applicable to potentially many stem cell systems, as well as diseases associated with stem cell maintenance including stem cell cancers, premature aging, and fertility decline.