The animal sperm nucleus is characterized by an extremely compacted organization of its DNA after the global replacement of histones with sperm-specific nuclear basic proteins, such as protamines. In the absence of DNA repair activity in the mature gamete, the integrity of the paternal genome is potentially challenged by the unique topological constraints exerted on sperm DNA. In addition, the maintenance of paternal DNA integrity during the rapid remodeling of sperm chromatin at fertilization has long been regarded as a maternal trait. However, little is known about the nature of the egg proteins involved in this essential aspect of zygote formation. We had previously characterized the unique phenotype of the classical Drosophila maternal effect mutant maternal haploid (mh), which specifically affects the integration of paternal chromosomes in the zygote. Here we show that MH is the fly ortholog of the recently identified human DVC1/Spartan protein, a conserved regulator of DNA damage tolerance. Like Spartan, MH protein is involved in the resistance to UV radiation and recruits the p97/TER94 segregase to stalled DNA replication forks in somatic cells. In the zygote, we found that the mh phenotype is consistent with perturbed or incomplete paternal DNA replication. Remarkably, however, the specific accumulation of MH in the male pronucleus before the first S phase suggests that this maternal protein is required to maintain paternal DNA integrity during nuclear decondensation or to set the paternal chromatin landscape in preparation of the first zygotic cycle.

The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

During spermiogenesis, histones are massively replaced with protamines. A previous report showed that Drosophila males homozygous for a genomic deletion covering several genes including the protamine-like genes Mst35Ba/b are surprisingly fertile. Here, we have precisely deleted the Mst35B locus by homologous recombination, and we confirm the dispensability of Mst35Ba/b for fertility.