Meiosis-specific gene functions and cell cycle control in Drosophila spermatocytes
Zusammenfassung der Projektergebnisse
Genome haploidization occurs in eukaryotes during meiosis in the context of sexual reproduction. In humans, in particular in females with increasing age, this fascinating but complex process is surprisingly error-prone, resulting in aneuploid zygotes that are usually aborted spontaneously before term with the exception of sex chromosome anomalies and certain trisomies (like trisomy 21, the cause of Down syndrome). Given that meiosis is a hallmark of eukaryote biology, evolutionary plasticity might appear unlikely. However, various aspects differ considerably between species. Therefore, findings obtained in the experimentally most accessible systems, i.e., in budding and fission yeast, are not necessarily valid for all other eukaryotes. In complex multicellular eukaryotes (animals and plants), the elucidation of the molecular mechanisms that govern genome haploidization remains a challenge as access to the meiotic stages is rather limited, precluding many biochemical approaches as well as efficient in vivo imaging. With our project, we intended to improve experimental accessibility in an animal by exploiting advantages of the model organism Drosophila melanogaster. We were able to develop transgenic RNA interference (RNAi) with greatly improved efficiency during the meiotic stages. GAL4-dependent expression of small hairpin micro RNAs (shmiRs) from transgenes generated with the VALIUM20 or VALIUM22 vector could be shown to result in efficient gene knock-down in spermatocytes in the majority of the targeted genes. VALIUM20/22-mediated RNAi is therefore not just effective in the female germline, as shown before, but also in males. Male meiosis is of interest in particular also because of its predicted advantages for application of the powerful method of in vivo time lapse imaging for phenotypic analyses. Indeed, we were able to develop an imaging protocol that is fully compatible with normal progression through both meiotic divisions with temporal and spatial resolution sufficient for complete tracking of individual centromeres. In addition, we have generated GAL4 driver lines that extend genetic manipulation to late spermatocytes and have established the functionality of targeted degradation of GFP fusion proteins (DeGradFP) in testis. Using these toolkit extensions, we have been able to characterize the function of centromere proteins (Cenp-A/Cid, Cenp-C, cal1) during meiosis. Our findings indicate that the loading of Cenp-A/Cid which acts at the top of the centromere/kinetochore assembly pathway during meiosis is mechanistically distinct from that operating during mitosis. While loading occurs during G1 when cells progress through the mitotic cell cycle, it happens during G2 in case of meiosis. Moreover, we were able to demonstrate that Cenp-A/Cid, a centromere-specific histone H3 variant, is not eliminated from chromatin during spermiogenesis in contrast to the bulk of nucleosomes and the other centromere proteins. Experimental alteration of the levels of Cenp-A/Cid in sperm demonstrated that this centromere protein is an essential component of the epigenetic centromere mark on paternal chromosomes. Moreover, it exerts quantitative control over centromeric Cenp-A/Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses. In addition, our methodological progress also allowed a functional characterization of genes that appeared to have a meiosis-specific function based on mining of transcriptome data. Thereby a Cenp-B- like gene could be identified as a novel member of the class of meiotic-arrest genes which control the transcription of a large set of genes required for meiosis and spermiogenesis.
Projektbezogene Publikationen (Auswahl)
- 2012. Transgenerational propagation and quantitative maintenance of paternal centromeres depends on Cid/Cenp-A presence in Drosophila sperm. PLoS Biol. 10(12):e1001434
Raychaudhuri, N., Dubruille, R., Orsi, G.A., Bagheri, H.C., Loppin, B., Lehner, C.F.
