Cells are required to divide and organize into tissues. One method of tissue homeostasis is the utilization of adult stem cells that are able to replace cells by undergoing asymmetric cell division (ACD) to produce a renewed stem cell and a differentiating cell. Disruptions to this division can lead to cancers or tissue degeneration. Although ACD is a vital part of tissue homeostasis, the underlying mechanisms involved in specifying two unique cell identities during this process are unknown. Previous work studying the epigenetic mechanisms of ACD in the Drosophila male germline found that, by labeling old and new histones distinctively, old H3 histones are retained in the renewed stem cell, whereas new H3 histones are preferentially segregated to the differentiating cell. To investigate whether asymmetric histone inheritance is an important mechanism of ACD, I will study histone inheritance in the intestinal stem cells (ISCs) of Drosophila. The ISCs undergo ACD to produce two differentiated cell types, enterocyte and enteroendocrine cells. The ISCs are also able to undergo symmetric cell division (SCD) to produce two ISCs. The ISC lineage is ideal to study histone inheritance as it is high throughput as there are many ISCs in one intestine, the ISC is the only cell in the lineage to undergo mitosis, and transgenes can be expressed in only the progenitor cells. I will take advantage of a two-color transgenic system to label old and new histones differentially and track their segregation during ISC division. My preliminary data suggest that H3 histones are segregated asymmetrically during the ACD of ISCs, while H3 histones are segregated symmetrically during SCD. In contrast, H2A histones are segregated symmetrically in both ACD and SCD. This suggests that H3 may be important for establishing different cell identities. To investigate the cellular and molecular mechanisms of histone inheritance, I will first look at old and new histone deposition in recently replicated sister chromatids and then study the segregation of the sister chromatids during mitosis. I will use a chromatin fiber technique to study the incorporation of histones following DNA replication in ISCs. I will then analyze components of the mitotic machinery that recognizes and segregates these epigenetically distinct sister chromatids during cell division. Further, I will investigate a mutation in the tail of the H3 histone, which has been previously found to cause aberrant histone inheritance and cellular defects such as tumors and tissue degeneration in the Drosophila germline. After characterizing the mechanism of H3 inheritance during cell division, I will investigate how H3 inheritance influences cell identity. To study this, I will examine the chromatin context at gene loci that are important for cell identity to determine if different chromatin contexts are established and inherited through cell division. I will also investigate gene expression in a wildtype and mutant H3 background to determine if mis-inheritance of H3 causes aberrant gene expression. This will provide understanding of how histones affect cell identity and the roles they may play in diseases such as cancer. The excellent research and training at Johns Hopkins University and in the Chen laboratory will allow me to both complete the proposed research and develop the skills necessary to pursue a career in academia.
Stem cells are required to undergo asymmetric cell division to provide a self-renewed stem cell and a differentiating daughter cell. Differential inheritance of histones is an important epigenetic mechanism for determining the two different cell identities that arise from the asymmetric cell division of the Drosophila germline stem cell. This proposal aims to investigate histone inheritance as a general mechanism of asymmetric cell division by studying it in the Drosophila intestinal stem cell, as well as to study the downstream effects of histone inheritance on gene expression and cell identity. 1
