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Abstract

The adult Drosophila midgut is an excellent model, in which to address how stem cell identity is defined and maintained, due to its simple structure, powerful genetics and similarity to the mammalian intestine. The epithelium turnover is carried out by multipotent intestinal stem cells (ISCs), which proliferate throughout life, renewing and generating transient committed cells called enteroblasts (EBs), which differentiate either into enterocytes (ECs) or enteroendocrine cells (EEs). The regulation for the progression from ISC to a terminally differentiated cell includes epigenetic mechanisms, however little is known about it in this specific stem cell system. In this study, I analyzed the distribution pattern of the histone H3 variant CENP-A in the adult Drosophila midgut. CENP-A is the epigenetic mark of centromeres, which identify the specific chromatin regions that mediates spindle attachment during chromosome segregation. Even though centromeres orchestrate chromosome inheritance, their positions on chromosomes are primarily specified epigenetically rather than by a specific DNA sequence in multicellular organisms. Employing different strategies, I found that CENP-A is asymmetrically inherited in cells of the midgut epithelium, where previously synthesized (‘old’) CENP-A is retained specifically in ISCs. Remarkably, long-term experiments revealed that CENP-A can persist in ISCs for more than 20 days. The stability and persistency of CENP-A supports the idea that CENP-A could act as an epigenetic mark responsible for regulating stem cell properties. Analyzing the distribution of this histone variant in somatic cells provided evidence that the asymmetric distribution of CENP-A is a mechanism specific of stem cells. In contrast to CENP-A, the histone variant H3.3 does not exhibit asymmetry during ISC division. CENP-A and its loading factor CAL1 have always been studied in the context of cell division. However, data from this study suggest that CENP-A may play a role in non-dividing cells as well. I could show that the depletion of inner kinetochore proteins in the non-dividing committed progenitor cells leads to the loss of these cell types, indicating that CENP-A and CAL1 are important for EBs maintenance and differentiation. ECs also seem to be affected by the depletion of kinetochore proteins, ECs undergo endocycles, that are also characteristic for salivary glands, follicle cells and ovarian nurse cells. Cells of salivary glands lacking CAL1 failed to undergo endoreduplication and correct S-phase progression. Taken all together, I propose a novel role of the histone H3 variant CENP-A in stemness, by which it contributes to the maintenance of intestinal stem cell identity. Furthermore, CENP-A and other inner kinetochore proteins are also important in non-dividing differentiated cells. Specifically, I identified CAL1 as a possible regulator of endocycle progression.