Versager is expressed at the histone-to-protamine transition during spermiogenesis and is required for embryonic chromosome transmission in Drosophila melanogaster

Abstract

Background: Chromatin remodeling is one of the most intriguing features of spermiogenesis, during which nuclei undergo drastic morphological changes leading to extensive chromatin compaction. Genetic and cytological accessibility make Drosophila melanogaster a powerful model to study this process. In fruit flies, paternal histones are largely replaced with sperm specific nuclear basic proteins in a highly coordinated manner. This remodeling is essential not only for sperm function but also for proper behavior of paternal chromosomes in the embryo. Our understanding of the changes associated with sperm chromatin and their role in embryonic chromosome behavior is incomplete, and will depend on the identification and characterization of additional components. One such newly identified gene, versager (vrs), is described here. Methods: Chromosome transmission was genetically monitored from vrs Z2566 males using chromosomespecific visible mutations. Recombination and deletion mapping was used to localize the mutation, and DNA sequencing was used to identify the causative lesion. Both in vivo RNAi expression knockdown and rescue by transgene expression of EGFP-tagged protein were used to verify the gene identity. The developmental expression pattern of Vrs was defined based on the EGFP signal in testis relative to RFP-tagged H2Av expression. Behavior of DAPI-stained chromosomes in early embryos from vrs Z2566 males was examined using confocal microscopy. Results: Genetic observations indicated that vrsZ2566 is required in males for high fidelity transmission of paternally derived chromosomes. DNA sequencing revealed that the vrs Z2566 mutation is a missense mutation in Celera predicted gene CG5538 and results in a D2V amino acid substitution. This residue was found to be conserved in Drosophila species and related Diptera. RNAi knockdown of vrs resulted in paternal-effect chromosome loss, and a vrs + -EGFP transgene fully rescued the mutant phenotype. Confocal microscopy of testis revealed nuclear localization of Vrs-EGFP specifically at the canoe stage of spermiogenesis, overlapping with the time of removal of H2Av-RFP at the histone-to-protamine transition. Examination of early stage embryos revealed micronuclei, isolated chromosomes and bridges indicative of chromosome loss events during the first three divisions. Approximately a quarter of later stage embryos arrested with an abnormal number of metaphase and fragmented nuclei. Conclusions: A novel sperm-specific paternal-effect gene, vrs, was identified that is expressed at the histone-to-protamine transition and is important for embryonic chromosome behavior and development. The histone-to-protamine transition may be a developmental period sensitive to perturbations that may lead to embryonic mitotic errors, aneuploidy and developmental arrest.

Histone removal in sperm protects paternal chromosomes from premature division at fertilization

“…Paternal effect mutants represent a rare genetic resource to investigate sperm functions that are specifically required for the integration of paternal chromosomes in the zygote (14)(15)(16)(17)(18). The pal mutant, which was isolated more than fifty years ago, has been known to induce sporadic loss of paternal chromosomes at the onset of embryonic development (14,19,20), but the origin of this phenotype is unknown.…”