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

The global replacement of histones with protamines in sperm chromatin is widespread in animals, including insects, but its actual function remains enigmatic. We show that in the Drosophila paternal effect mutant paternal loss (pal), sperm chromatin retains germline histones H3 and H4 genome wide without impairing sperm viability. However, after fertilization, pal sperm chromosomes are targeted by the egg chromosomal passenger complex and engage into a catastrophic premature division in synchrony with female meiosis II. We show that pal encodes a rapidly evolving transition protein specifically required for the eviction of (H3-H4)2 tetramers from spermatid DNA after the removal of H2A-H2B dimers. Our study thus reveals an unsuspected role of histone eviction from insect sperm chromatin: safeguarding the integrity of the male pronucleus during female meiosis.

APOLLO, a testis-specific Drosophila ortholog of importin-4, mediates the loading of protamine-like protein Mst77F into sperm chromatin.

DNA in sperm is packed with small, charged proteins termed SNBPs (sperm nuclear basic proteins), including mammalian and Drosophila protamines. During spermiogenesis, somatic-type chromatin is taken apart and replaced with sperm chromatin in a multistep process leading to an extraordinary condensation of the genome. During fertilization, the ova face a similarly challenging task of SNBP eviction and reassembly of nucleosome-based chromatin. Despite its importance for the animal life cycle, sperm chromatin metabolism, including the biochemical machinery mediating the mutual replacement of histones and SNBPs, remains poorly studied. In Drosophila, Mst77F is one of the first SNBPs loaded into the spermatid nuclei. It persists in mature spermatozoa and is essential for sperm compaction and male fertility. Here, by using in vitro biochemical assays, we identify chaperones that can mediate the eviction and loading of Mst77F on DNA, thus facilitating the interconversions of chromatin forms in the male gamete. Unlike NAP1 and TAP/p32 chaperones that disassemble Mst77F-DNA complexes, ARTEMIS and APOLLO, orthologs of mammalian importin-4 (IPO4), mediate the deposition of Mst77F on DNA or oligonucleosome templates, accompanied by the dissociation of histone-DNA complexes. In vivo, a mutation of testis-specific Apollo brings about a defect of Mst77F loading, abnormal sperm morphology, and male infertility. We identify IPO4 ortholog APOLLO as a critical component of sperm chromatin assembly apparatus in Drosophila. We discover that in addition to recognized roles in protein traffic, a nuclear transport receptor (IPO4) can function directly in chromatin remodeling as a dual, histone- and SNBP-specific, chaperone.

Biophysical ordering transitions underlie genome 3D re-organization during cricket spermiogenesis.

Spermiogenesis is a radical process of differentiation whereby sperm cells acquire a compact and specialized morphology to cope with the constraints of sexual reproduction while preserving their main cargo, an intact copy of the paternal genome. In animals, this often involves the replacement of most histones by sperm-specific nuclear basic proteins (SNBPs). Yet, how the SNBP-structured genome achieves compaction and accommodates shaping remain largely unknown. Here, we exploit confocal, electron and super-resolution microscopy, coupled with polymer modeling to identify the higher-order architecture of sperm chromatin in the needle-shaped nucleus of the emerging model cricket Gryllus bimaculatus. Accompanying spermatid differentiation, the SNBP-based genome is strikingly reorganized as ~25nm-thick fibers orderly coiled along the elongated nucleus axis. This chromatin spool is further found to achieve large-scale helical twisting in the final stages of spermiogenesis, favoring its ultracompaction. We reveal that these dramatic transitions may be recapitulated by a surprisingly simple biophysical principle based on a nucleated rigidification of chromatin linked to the histone-to-SNBP transition within a confined nuclear space. Our work highlights a unique, liquid crystal-like mode of higher-order genome organization in ultracompact cricket sperm, and establishes a multidisciplinary methodological framework to explore the diversity of non-canonical modes of DNA organization.

Essential and recurrent roles for hairpin RNAs in silencing de novo sex chromosome conflict in Drosophila simulans.

Meiotic drive loci distort the normally equal segregation of alleles, which benefits their own transmission even in the face of severe fitness costs to their host organism. However, relatively little is known about the molecular identity of meiotic drivers, their strategies of action, and mechanisms that can suppress their activity. Here, we present data from the fruitfly Drosophila simulans that address these questions. We show that a family of de novo, protamine-derived X-linked selfish genes (the Dox gene family) is silenced by a pair of newly emerged hairpin RNA (hpRNA) small interfering RNA (siRNA)-class loci, Nmy and Tmy. In the w[XD1] genetic background, knockout of nmy derepresses Dox and MDox in testes and depletes male progeny, whereas knockout of tmy causes misexpression of PDox genes and renders males sterile. Importantly, genetic interactions between nmy and tmy mutant alleles reveal that Tmy also specifically maintains male progeny for normal sex ratio. We show the Dox loci are functionally polymorphic within D. simulans, such that both nmy-associated sex ratio bias and tmy-associated sterility can be rescued by wild-type X chromosomes bearing natural deletions in different Dox family genes. Finally, using tagged transgenes of Dox and PDox2, we provide the first experimental evidence Dox family genes encode proteins that are strongly derepressed in cognate hpRNA mutants. Altogether, these studies support a model in which protamine-derived drivers and hpRNA suppressors drive repeated cycles of sex chromosome conflict and resolution that shape genome evolution and the genetic control of male gametogenesis.

Functional analysis of Wolbachia Cid effectors unravels cooperative interactions to target host chromatin during replication.

Wolbachia are common bacteria among terrestrial arthropods. These endosymbionts transmitted through the female germline manipulate their host reproduction through several mechanisms whose most prevalent form called Cytoplasmic Incompatibility -CI- is a conditional sterility syndrome eventually favoring the infected progeny. Upon fertilization, the sperm derived from an infected male is only compatible with an egg harboring a compatible Wolbachia strain, this sperm leading otherwise to embryonic death. The Wolbachia Cif factors CidA and CidB responsible for CI and its neutralization function as a Toxin-Antitoxin system in the mosquito host Culex pipiens. However, the mechanism of CidB toxicity and its neutralization by the CidA antitoxin remain unexplored. Using transfected insect cell lines to perform a structure-function analysis of these effectors, we show that both CidA and CidB are chromatin interactors and CidA anchors CidB to the chromatin in a cell-cycle dependent-manner. In absence of CidA, the CidB toxin localizes to its own chromatin microenvironment and acts by preventing S-phase completion, independently of its deubiquitylase -DUB- domain. Experiments with transgenic Drosophila show that CidB DUB domain is required together with CidA during spermatogenesis to stabilize the CidA-CidB complex. Our study defines CidB functional regions and paves the way to elucidate the mechanism of its toxicity.

Paternal transmission of the Wolbachia CidB toxin underlies cytoplasmic incompatibility.

Wolbachia are widespread endosymbiotic bacteria that manipulate the reproduction of arthropods through a diversity of cellular mechanisms. In cytoplasmic incompatibility (CI), a sterility syndrome originally discovered in the mosquito Culex pipiens, uninfected eggs fertilized by sperm from infected males are selectively killed during embryo development following the abortive segregation of paternal chromosomes in the zygote. Despite the recent discovery of Wolbachia CI factor (cif) genes, the mechanism by which they control the fate of paternal chromosomes at fertilization remains unknown. Here, we have analyzed the cytological distribution and cellular impact of CidA and CidB, a pair of Cif proteins from the Culex-infecting Wolbachia strain wPip. We show that expression of CidB in Drosophila S2R+ cells induces apoptosis unless CidA is co-expressed and associated with its partner. In transgenic Drosophila testes, both effectors colocalize in germ cells until the histone-to-protamine transition in which only CidB is retained in maturing spermatid nuclei. We further show that CidB is similarly targeted to maturing sperm of naturally infected Culex mosquitoes. At fertilization, CidB associates with paternal DNA regions exhibiting DNA replication stress, as a likely cause of incomplete replication of paternal chromosomes at the onset of the first mitosis. Importantly, we demonstrate that inactivation of the deubiquitylase activity of CidB does not abolish its cell toxicity or its ability to induce CI in Drosophila. Our study thus demonstrates that CI functions as a transgenerational toxin-antidote system and suggests that CidB acts by poisoning paternal DNA replication in incompatible crosses.

Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain.

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.

Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system.

Segregation Distorter (SD) is a male meiotic drive system in Drosophila melanogaster. Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD+ chromosome. It is well established that distortion results from an interaction between Sd, the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp, on the SD+ chromosome. However, the molecular and cellular mechanisms leading to post-meiotic SD+ sperm elimination remain unclear. Here we show that SD/SD+ males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD+ spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD+ genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. Our analyses of different SD+ chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. We propose that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization.

Histone Variants: The Nexus of Developmental Decisions and Epigenetic Memory.

Nucleosome dynamics and properties are central to all forms of genomic activities. Among the core histones, H3 variants play a pivotal role in modulating nucleosome structure and function. Here, we focus on the impact of H3 variants on various facets of development. The deposition of the replicative H3 variant following DNA replication is essential for the transmission of the epigenomic information encoded in posttranscriptional modifications. Through this process, replicative H3 maintains cell fate while, in contrast, the replacement H3.3 variant opposes cell differentiation during early embryogenesis. In later steps of development, H3.3 and specialized H3 variants are emerging as new, important regulators of terminal cell differentiation, including neurons and gametes. The specific pathways that regulate the dynamics of the deposition of H3.3 are paramount during reprogramming events that drive zygotic activation and the initiation of a new cycle of development.

The Enigma of Centriole Loss in the 1182-4 Cell Line.

The Drosophilamelanogaster cell line 1182-4, which constitutively lacks centrioles, was established many years ago from haploid embryos laid by females homozygous for the maternal haploid (mh) mutation. This was the first clear example of animal cells regularly dividing in the absence of this organelle. However, the cause of the acentriolar nature of the 1182-4 cell line remained unclear and could not be clearly assigned to a particular genetic event. Here, we detail historically the longstanding mystery of the lack of centrioles in this Drosophila cell line. Recent advances, such as the characterization of the mh gene and the genomic analysis of 1182-4 cells, allow now a better understanding of the physiology of these cells. By combining these new data, we propose three reasonable hypotheses of the genesis of this remarkable phenotype.

The Lid/KDM5 histone demethylase complex activates a critical effector of the oocyte-to-zygote transition.

Following fertilization of a mature oocyte, the formation of a diploid zygote involves a series of coordinated cellular events that ends with the first embryonic mitosis. In animals, this complex developmental transition is almost entirely controlled by maternal gene products. How such a crucial transcriptional program is established during oogenesis remains poorly understood. Here, we have performed an shRNA-based genetic screen in Drosophila to identify genes required to form a diploid zygote. We found that the Lid/KDM5 histone demethylase and its partner, the Sin3A-HDAC1 deacetylase complex, are necessary for sperm nuclear decompaction and karyogamy. Surprisingly, transcriptomic analyses revealed that these histone modifiers are required for the massive transcriptional activation of deadhead (dhd), which encodes a maternal thioredoxin involved in sperm chromatin remodeling. Unexpectedly, while lid knock-down tends to slightly favor the accumulation of its target, H3K4me3, on the genome, this mark was lost at the dhd locus. We propose that Lid/KDM5 and Sin3A cooperate to establish a local chromatin environment facilitating the unusually high expression of dhd, a key effector of the oocyte-to-zygote transition.

The hpRNA/RNAi Pathway Is Essential to Resolve Intragenomic Conflict in the Drosophila Male Germline.

Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. Here, we reveal that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, we generated D. simulans null mutants of Dcr-2 and AGO2. Strikingly, these core RNAi knockouts massively derepress Dox and MDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, our data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species.

ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization.

BACKGROUND: Anti-Silencing Factor 1 (ASF1) is a conserved H3-H4 histone chaperone involved in both Replication-Coupled and Replication-Independent (RI) nucleosome assembly pathways. At DNA replication forks, ASF1 plays an important role in regulating the supply of H3.1/2 and H4 to the CAF-1 chromatin assembly complex. ASF1 also provides H3.3-H4 dimers to HIRA and DAXX chaperones for RI nucleosome assembly. The early Drosophila embryo is an attractive system to study chromatin assembly in a developmental context. The formation of a diploid zygote begins with the unique, genome-wide RI assembly of paternal chromatin following sperm protamine eviction. Then, within the same cytoplasm, syncytial embryonic nuclei undergo a series of rapid, synchronous S and M phases to form the blastoderm embryo. Here, we have investigated the implication of ASF1 in these two distinct assembly processes. RESULTS: We show that depletion of the maternal pool of ASF1 with a specific shRNA induces a fully penetrant, maternal effect embryo lethal phenotype. Unexpectedly, despite the depletion of ASF1 protein to undetectable levels, we show that asf1 knocked-down (KD) embryos can develop to various stages, thus demonstrating that ASF1 is not absolutely required for the amplification of cleavage nuclei. Remarkably, we found that ASF1 is required for the formation of the male pronucleus, although ASF1 protein does not reside in the decondensing sperm nucleus. In asf1 KD embryos, HIRA localizes to the male nucleus but is only capable of limited and insufficient chromatin assembly. Finally, we show that the conserved HIRA B domain, which is involved in ASF1-HIRA interaction, is dispensable for female fertility. CONCLUSIONS: We conclude that ASF1 is critically required to load H3.3-H4 dimers on the HIRA complex prior to histone deposition on paternal DNA. This separation of tasks could optimize the rapid assembly of paternal chromatin within the gigantic volume of the egg cell. In contrast, ASF1 is surprisingly dispensable for the amplification of cleavage nuclei, although chromatin integrity is likely compromised in KD embryos.

Does pupal communication influence Wolbachia-mediated cytoplasmic incompatibility?

Wolbachia are widespread endosymbiotic bacteria found in terrestrial arthropods and filarial nematodes [1]. In insects, Wolbachia generally rely on diverse strategies to manipulate their host's reproduction and favor their own vertical transmission through infected eggs [2]. One such mechanism is a sterility syndrome called 'cytoplasmic incompatibility'. Cytoplasmic incompatibility occurs at fertilization, when a spermatozoon from a Wolbachia-infected male fertilizes an uninfected egg. In this case, sperm-derived chromosomes fail to separate normally at the first zygotic division, thus preventing the development of a diploid embryo [3]. Moreover, the presence of Wolbachia in females rescues the integration of paternal chromosomes in the zygote and allows the development of a viable, infected individual. Although the molecular basis of cytoplasmic incompatibility is still unknown, a current model implies the existence of Wolbachia-induced reversible modifications on sperm DNA or chromatin that must be eliminated or neutralized shortly after fertilization by rescuing Wolbachia factors present in infected eggs [4]. In a recent Current Biology paper [5], Stéphanie Pontier and François Schweisguth recently challenged this model by proposing that Wolbachia perturbs a pheromone-based communication between male and female pupae in Drosophila melanogaster and Drosophila simulans, which controls the "compatibility range" of male and female gametes. However, we fail to detect any influence of pupal communication on cytoplasmic incompatibility in Drosophila simulans as well as in the parasitoid wasp Nasonia vitripennis. Our results thus question the robustness of their model.

Unlocking sperm chromatin at fertilization requires a dedicated egg thioredoxin in Drosophila.

In most animals, the extreme compaction of sperm DNA is achieved after the massive replacement of histones with sperm nuclear basic proteins (SNBPs), such as protamines. In some species, the ultracompact sperm chromatin is stabilized by a network of disulfide bonds connecting cysteine residues present in SNBPs. Studies in mammals have established that the reduction of these disulfide crosslinks at fertilization is required for sperm nuclear decondensation and the formation of the male pronucleus. Here, we show that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization. In dhd mutant eggs, the sperm nucleus fails to decondense and the replacement of SNBPs with maternally-provided histones is severely delayed, thus preventing the participation of paternal chromosomes in embryo development. We demonstrate that DHD localizes to the sperm nucleus to reduce its disulfide targets and is then rapidly degraded after fertilization.

The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin.

In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. Here, we show that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35Ba/b. We demonstrate that Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals.

Rapid evolution of a Y-chromosome heterochromatin protein underlies sex chromosome meiotic drive.

Sex chromosome meiotic drive, the non-Mendelian transmission of sex chromosomes, is the expression of an intragenomic conflict that can have extreme evolutionary consequences. However, the molecular bases of such conflicts remain poorly understood. Here, we show that a young and rapidly evolving X-linked heterochromatin protein 1 (HP1) gene, HP1D2, plays a key role in the classical Paris sex-ratio (SR) meiotic drive occurring in Drosophila simulans Driver HP1D2 alleles prevent the segregation of the Y chromatids during meiosis II, causing female-biased sex ratio in progeny. HP1D2 accumulates on the heterochromatic Y chromosome in male germ cells, strongly suggesting that it controls the segregation of sister chromatids through heterochromatin modification. We show that Paris SR drive is a consequence of dysfunctional HP1D2 alleles that fail to prepare the Y chromosome for meiosis, thus providing evidence that the rapid evolution of genes controlling the heterochromatin structure can be a significant source of intragenomic conflicts.

The intimate genetics of Drosophila fertilization.

The union of haploid gametes at fertilization initiates the formation of the diploid zygote in sexually reproducing animals. This founding event of embryogenesis includes several fascinating cellular and nuclear processes, such as sperm-egg cellular interactions, sperm chromatin remodelling, centrosome formation or pronuclear migration. In comparison with other aspects of development, the exploration of animal fertilization at the functional level has remained so far relatively limited, even in classical model organisms. Here, we have reviewed our current knowledge of fertilization in Drosophila melanogaster, with a special emphasis on the genes involved in the complex transformation of the fertilizing sperm nucleus into a replicated set of paternal chromosomes.