Chromatin Structure Changes During Terminal Differentiation and Cell Cycle Exit in Drosophila melanogaster

Abstract

During development, the number of cell divisions must be precisely controlled in order to produce tissues of the correct shape, composition and size. The majority of cells complete their final cell cycle during a process called terminal differentiation, where cells acquire cell type specific characteristics. Most terminally differentiated cells will remain in a post-mitotic or G0 state permanently to carry out critical physiological functions in tissues and organs. The enforcement of cell cycle exit is thought to be critical for proper differentiation, but how these events are coordinated in most tissues remains unclear. Chromatin accessibility and organization plays a critical role in regulating gene expression during differentiation and changes in chromatin organization also occur upon entry into G0. In my thesis research I addressed how chromatin organization and accessibility changes during terminal differentiation and cell cycle exit in the Drosophila melanogaster (fruit fly) wing. To examine the relationship between cell cycle exit and chromatin structure during terminal differentiation, I characterized the temporal changes in chromatin accessibility and gene expression during the process of terminal differentiation in the wing. This revealed changes in chromatin accessibility and gene expression that are coordinated with the transition from a proliferating to postmitotic state. To identify which changes are a consequence of cell cycle exit, I genetically disrupted cell cycle exit and examined the effects on chromatin accessibility and gene expression. This uncovered mutual cross-talk between a subset of genes in the wing terminal differentiation program and the cell cycle machinery. However, most chromatin changes including those at cell cycle genes, appear to be developmentally controlled in a manner independent of cell cycling status. Higher order chromatin organization such as the clustering of heterochromatin in the nucleus is also impacted by cell cycle exit and terminal differentiation. I found that heterochromatin clusters as cells exit the cell cycle and terminally differentiate. Heterochromatin associated modifications have been implicated in the silencing of cell cycle genes and facilitating G0. I rigorously tested this model and found that compromising heterochromatin-dependent gene silencing does not disrupt cell cycle exit. Instead, delaying or preventing cell cycle exit disrupts heterochromatin clustering and globally alters chromatin modifications, revealing that heterochromatin clustering during terminal differentiation is a consequence of cell cycle exit, rather than differentiation.