The Function and Regulation of Drosophila melanogaster Centromeric Chromatin in Mitosis, Meiosis and Cancer

Centromeres are regions of eukaryotic chromosomes essential for faithful segregation of DNA during all types of cell divisions. In most eukaryotes, centromere identity is defined and maintained epigenetically through cell generations by the presence of the centromere-specific histone H3 variant CENP-A, termed CID in the fruit fly Drosophila melanogaster. How CENP-A is incorporated exclusively at centromeres and reproducibly propagated during the cell cycle is a key question in the centromere field. Improper regulation of CENP-A assembly leads to the formation of ectopic centromeres, aberrant segregation of chromosomes, and aneuploidy, which can culminate in cell death or contribute to tumorigenesis. Recent studies have discovered cell cycle mechanisms and factors regulating the maintenance and assembly of CENP-A at centromeres during mitosis, and have paved the way for investigations into the mechanisms that specify centromere identity, function and regulation in the animal./nMeiosis is an essential part of the reproductive cycle in most eukaryotes that encompasses two phases of chromosome segregation, defects in which result in aneuploid eggs, sperm and the resulting zygotes. The role, regulation, and cell cycle timing of CENP-A assembly during meiosis and mitosis in animal tissues was previously unknown. To understand the dynamics and regulation of CENP-A assembly in somatic mitotic tissues and during meiosis, we investigated CENP-ACID assembly dynamics during gametogenesis and development in Drosophila. We tracked CENP-ACID levels at centromeres in the meiotic divisions during spermatogenesis and mitotic divisions in the developing larval brain. We found that CID assembly at centromeres occurs at different cell cycle phases in mitotic divisions in the larval brain and meiotic divisions in male and female gametogenesis. This timing of mitotic and meiotic CID assembly differs from previous observations in cultured cells and embryos. We also demonstrated that CID is maintained on mature sperm despite the extensive chromatin remodeling and histone removal that occurs during protamine exchange./nCentromeric proteins are commonly misregulated in many types of human cancers, and higher levels correlate with poor prognosis and response to treatment. Because excess CENP-A can cause chromosome mis-segregation via the formation of ectopic kinetochores or endogenous centromere dysfunction, overexpression of centromere proteins may be a mechanism for the creation or maintenance of chromosomal instability and aneuploidy in cancer cells. Using a Drosophila model of human glioblastoma, which recapitulates cancer growth and metastasis through activation of the conserved EGFR/PI3K pathways in fly glial cells, we determined the effect of overexpression of CENP-ACID and its chaperone/assembly factor, the HJURP functional homolog CAL1, on cancer phenotypes and progression. CENP-ACID or CAL1 overexpression in combination with PI3K activation, which alone does not induce overproliferation, was sufficient for hyperplastic growth. We also investigated the effect of centromere protein misregulation on the genome. CENP-ACID overexpression resulted in increased genome instability and aneuploidy, localized to specific euchromatic sites, and altered gene expression./nThese studies provide insight into the role and regulation of centromeric proteins in the animal, and are a foundation for further investigations into the regulation of centromeric chromatin assembly and maintenance. Additionally, these studies point towards the need to study the role of CENP-A and HJURP in human cancers with the goal of identifying new diagnostic and treatment targets.

Advisor: Karpen, Gary