Cell cycle regulation of centromere assembly in Drosophila male meiosis

Abstract

Centromeres are chromosomal sites required for faithful chromosome segregation. CENP-A is the histone H3 variant that demarcates centromeres from bulk chromatin. Errors in centromere assembly result in segregation defects that can lead to genome instability, sterility and genetic disorders. In Drosophila, the CENP-A assembly partners CENP-C and CAL1 assist in constructing a functional centromere that provides a platform for kinetochore assembly. In this study, we exploit Cenp-CZ3-4375 and cal12k32 mutants to explore the role of CENP-C and CAL1 in CENP-A assembly in male meiosis (Chapter 3). We find that levels of CENP-A are reduced in Cenp-CZ3-4375 and cal12k32 mutants. We report for the first time that CENP-A is sequestered in the nucleolus in wild type spermatocytes. We further show a correlation between nucleolar integrity and CENP-A levels. Nucleolar disruption and reduced global RNA transcription correlates to reduced centromeric CENP-A in Cenp-CZ3-4375 spermatocytes and segregation defects including lagging chromosomes and decondensed chromatin in spermatids. In Chapter 4 and 5 we characterise cell cycle regulation of meiotic CENP-A assembly given its temporal disconnect from mitosis. Meiotic CENP-A assembly is coupled to high Cdk activity, which in mitosis, inhibits assembly. We report that Cdk1/2 inhibition in early prophase I spermatocytes stimulates CENP-A incorporation to centromeres. We find that Cdk1/2 binding partners CYCA and CYCB localise to meiotic centromeres and that overexpression of stable CYCA or CYCB compromises CENP-A assembly. Testis-specific RNAi and site directed mutagenesis studies implicate CAL1 as the potential substrate through which CYCA or CYCB associated kinase activity mediates this inhibitory regulation. In Chapter 6 we uncover a single phosphorylation site on CAL1 that is important for Cdk mediated regulation of meiotic CENP-A assembly. We conclude that perturbation of CENP-C or CAL1 has adverse effects on centromere assembly and ultimately chromosome segregation and fertility.