Investigating the mechanisms and generality of asymmetric histone inheritance in multiple stem cell systems

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 DESCRIPTION (provided by applicant): Epigenetic phenomena create heritable changes in gene expression or function without altering primary DNA sequences. Epigenetic information directs cells with the same genome to take on distinct morphologies and different functions necessary for proper development. Many types of stem cells undergo asymmetric cell divisions to give rise to daughter cells with distinct cell fates: one that retains stem cell identity and another that differentiates. A long-standing question has been how the epigenetic information of a stem cell is transferred to the daughter cells. Using the Drosophila male germline stem cell (GSC) lineage, recent studies in our laboratory have shown that epigenetic information is inherited asymmetrically during asymmetric stem cell division (ACD). My research addresses two fundamental questions raised by this finding. (1) What are the molecular mechanisms used to ensure proper passage of epigenetic information during asymmetric cell division? (2) What other stem cell systems demonstrate asymmetric inheritance of epigenetic information? Using an innovative dual-color histone labelling method, our laboratory has been able to visualize the segregation patterns of the H3 histones during GSC ACD. By selectively labeling preexisting vs. newly synthesized H3, we have been able to demonstrate that preexisting histones preferentially segregate to the daughter cell fated to become the GSC, whereas newly synthesized H3 preferentially segregate to the daughter fated to become the gonialblast (GB). Histone H3.3, which is incorporated in a replication manner, shows a symmetric mode of inheritance. This finding, coupled with the fact that a majority of canonical histones are incorporated during DNA replication, suggests that the process of DNA replication may play a novel role in helping to establish asymmetric histone distribution observed in H3. We hypothesize that certain aspects of replication machinery are involved in retention and deposition of preexisting histones during DNA replication. Using an image-based protein-protein proximity ligation assay (PLA), we will test potential interactions between H3 (or modified H3) and DNA replication machinery components. We will test the functional role of key molecules identified in the PLA using an RNAi screen. Using the dual-color histone labelling method, I will explore the mode of inheritance exhibited by histone proteins H1, H2A, H2B and H4 to better understand what components of the chromatosome may play a part in establishing the epigenetic identity of a cell following ACD. I will also adapt the histone labelling method to labe histone proteins in the GSC of Drosophila ovary to determine if asymmetric histone distribution is present in multiple stem cell systems Improving our understanding of the molecular mechanisms governing epigenetic inheritance in stems cells will have significant implications on the fields of stem cell biology and regenerative medicine.