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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 exploited confocal, electron and super-resolution microscopy observations, coupled with polymer modeling simulations to identify the higher-order architecture of sperm chromatin in the needle-shaped nucleus of the emerging model cricket Gryllus bimaculatus. Accompanying spermatid differentiation and shaping, the SNBP-based genome was strikingly reorganized as ~25nm-thick fibers orderly coiled along the elongated nucleus axis. This chromatin spool was further found to achieve large-scale helical twisting in the final stages of spermiogenesis, favoring its ultracompaction. Through a combination of microscopy observations and polymer simulations, we revealed 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 completely distinct from nucleosomal chromatin, and establishes a multidisciplinary methodological framework to explore the diversity of non-canonical modes of DNA organization.SIGNIFICANCE STATEMENTAnimal sperm cells are highly compact and atypically shaped compared to other cell types. How DNA is packaged and organized in the 3D space of sperm cell nuclei to cope with these constraints is poorly understood. In this work, we identified an original and elegant solution to this problem in crickets, whereby DNA fibers orderly spool and twist to fit into ultracompact, needle-shaped sperm cells. To understand this reorganization, we modeled DNA fibers in the nucleus as polymers and found that a relatively simple mechanism through which fibers become more rigid bit by bit can largely recapitulate our observations. Our multidisciplinary work highlights a simple solution to compact DNA to extreme levels in specialized nuclei.

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., Author summary Multicellular development depends on a tight control of gene expression in each cell type. This relies on the coordinated activities of nuclear proteins that interact with DNA or its histone scaffold to promote or restrict gene transcription. For example, we previously showed that the histone modifying enzymes Lid and Sin3A/Rpd3 are required in Drosophila ovaries for the massive expression of deadhead (dhd), a gene encoding for a thioredoxin that is essential for fertility. In this paper, we have further identified two additional dhd regulators, Snr1 and Mod(mdg4) and dissected the mechanism behind hyperactivation of this gene. Using the epigenomic profiling method Cut&Run with a dedicated data analysis approach, we unexpectedly found that dhd is embedded in an unusual chromatin mini-domain featuring repressive histone modifications H3K27me3 and H3K9me3 and flanked by two regulatory elements. However, we further showed that Lid, Sin3A, Snr1 and Mod(mdg4) behave like obligatory activators of dhd independently of this mini-domain. Our study unveils how multiple broad-acting epigenomic effectors operate in non-canonical manners to ensure a critical and specialized gene activation event. These findings challenge our knowledge on these regulatory mechanisms and their roles in development and pathology.

International audience; 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.

Segregation Distorter(SD) is a male meiotic drive system inDrosophila melanogaster.Males heterozygous for a selfishSDchromosome rarely transmit the homologousSD+chromosome. It is well established that distortion results from an interaction betweenSd, the primary distorting locus on theSDchromosome and its target, a satellite DNA calledRsp,on theSD+chromosome. However, the molecular and cellular mechanisms leading to post-meioticSD+sperm elimination remain unclear. Here we show thatSD/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 otherSD/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 differentSD+chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of theRspsatellite. We propose that whenRspcopy 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.

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 Drosophila melanogaster 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.

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., Author summary Nuclear enzymes that add or remove epigenetic marks on histone tails potentially control gene expression by affecting chromatin structure and DNA accessibility. For instance, members of the KDM5 family of histone demethylases specifically remove methyl groups on the lysine 4 of histone H3, a mark generally correlated with gene expression. Lid (Little imaginal discs), the Drosophila KDM5, is essential for viability but is also required for female fertility. In this paper, we have found that the specific removal of Lid in developing oocytes perturbs the decompaction of the sperm nucleus at fertilization and the integration of paternal chromosomes in the zygote. Sperm nuclear decompaction normally requires the presence of a small redox protein called Deadhead (Dhd), which is massively expressed at the end of oogenesis. Strikingly, our analyses of ovarian transcriptomes revealed that the absence of Lid completely abolishes the expression of dhd. This direct functional link between a general histone modifier and the expression of an essential terminal effector gene represents a rare finding. We hope that our work will help understanding how histone demethylases function in controlling complex developmental transitions as well as cancer progression.

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.

Summary 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.

Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3-dependent intellectual disability.

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.

Centre de Ge ´ne´tique et de Physiologie Moleculaire etCellulaire,CNRSUMR5534,Universite´ ClaudeBernardLyon1,University of Lyon, Villeurbanne 69100, FranceSummaryThe animal sperm nucleus is characterized by an extremelycompacted organization of its DNA after the global replace-mentofhistoneswithsperm-specificnuclearbasicproteins,suchasprotamines[1,2].IntheabsenceofDNArepairactiv-ityinthematuregamete,theintegrityofthepaternalgenomeis potentially challenged by the unique topological con-straints exerted on sperm DNA [3]. In addition, the mainte-nance of paternal DNA integrity during the rapid remodelingofspermchromatinatfertilizationhaslongbeenregardedasa maternal trait [4]. However, little is known about the natureof the egg proteins involved in this essential aspect ofzygote formation [5, 6]. We had previously characterizedthe unique phenotype of the classical Drosophila maternaleffect mutant maternal haploid (mh), which specifically af-fects the integration of paternal chromosomes in the zygote[7]. Here we show that MH is the fly ortholog of the recentlyidentified human DVC1/Spartan protein, a conserved regu-lator of DNA damage tolerance [8–14]. Like Spartan, MH pro-tein isinvolved intheresistance toUVradiation andrecruitsthe p97/TER94 segregase to stalled DNA replication forks insomaticcells.Inthezygote,wefoundthatthemhphenotypeis consistent with perturbed or incomplete paternal DNAreplication.Remarkably,however,thespecificaccumulationof MH in the male pronucleus before the first S phase sug-gests that this maternal protein is required to maintainpaternal DNA integrity during nuclear decondensation orto set the paternal chromatin landscape in preparation ofthe first zygotic cycle.Results and DiscussionMolecular Identification of the maternal haploid GeneThe original mh

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., In many animals, sperm DNA compaction is achieved by disulfide bonds between the sperm nuclear proteins that replace the histones. Here, the authors provide evidence that Drosophila maternal thioredoxin Deadhead is required to unlock the sperm chromatin upon fertilization.

International audience; 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.

Comparative genome analysis has allowed the identification of various mechanisms involved in gene birth. However, understanding the evolutionary forces driving new gene origination still represents a major challenge. In particular, an intriguing and not yet fully understood trend has emerged from the study of new genes: many of them show a testis-specific expression pattern, which has remained poorly understood. Here we review the case of such a new gene, which involves a telomere-capping gene family inDrosophila. hiphopand its testis-specific paralogK81are critical for the protection of chromosome ends in somatic cells and male gametes, respectively. Two independent functional studies recently proposed that these genes evolved under a reproductive-subfunctionalization regime. The 2011 release of newDrosophilagenome sequences from themelanogastergroup of species allowed us to deepen our phylogenetic analysis of thehiphop/K81family. This work reveals an unsuspected dynamic of gene birth and death within the group, with recurrent duplication events through retroposition mechanisms. Finally, we discuss the plausibility of different evolutionary scenarios that could explain the diversification of this gene family.

International audience; A critical function of telomeres is to prevent the ligation of chromosome ends by DNA repair enzymes. In most eukaryotes, telomeric DNA consists in large arrays of G-rich tandem repeats that are recognized by DNA binding capping proteins. Drosophila telomeres are unusual as they lack short tandem repeats. However, Drosophila capping proteins can bind chromosome extremities in a DNA sequence-independent manner. This epigenetic protection of fly telomeres has been essentially studied in somatic cells where capping proteins such as HOAP or HP1 are essential in preventing chromosome end-to-end fusions. HipHop and K81 are two recently identified paralogous capping proteins with complementary expression patterns. While HipHop is involved in telomere capping in somatic cells, K81 has specialized in the protection of telomeres in post-meiotic male germ cells. Remarkably, K81 is required for the stabilization of HOAP and HP1 at telomeres during the massive paternal chromatin remodeling that occurs during spermiogenesis and at fertilization. We thus propose that the maintenance of capping proteins at Drosophila sperm telomeres is crucial for the transmission of telomere identity to the diploid zygote.

International audience; The Drosophila I-R type of hybrid dysgenesis is a sterility syndrome (SF sterility) associated with the mobilization of the I retrotransposon in female germ cells. SF sterility results from a maternal-effect embryonic lethality whose origin has remained unclear since its discovery about 40 years ago. Here, we show that meiotic divisions in SF oocytes are catastrophic and systematically fail to produce a functional female pronucleus at fertilization. As a consequence, most embryos from SF females rapidly arrest their development with aneuploid or damaged nuclei, whereas others develop as non-viable, androgenetic haploid embryos. Finally, we show that, in contrast to mutants affecting the biogenesis of piRNAs, SF egg chambers do not accumulate persistent DNA double-strand breaks, suggesting that I-element activity might perturb the functional organization of meiotic chromosomes without triggering an early DNA damage response.

The EMBO Workshop on Genomic Imprinting took place between 21 and 24 September 2008, in Singapore, and was organized by F. Berger, M. Bartolomei and R. Feil. This painting, entitled ‘Back of Telok Ayer Street 2007’ and painted by C. Ek Kay, was the image included in the meeting poster. ![][1] See Glossary for abbreviations used in this article The organizers of the European Molecular Biology Organization Workshop on Genomic Imprinting chose a truly trans‐kingdom selection of speakers; there was a strong representation of those working in plants and mammals, with a sprinkling of those studying flies and worms. This organismal diversity was layered further with talks on topics ranging from germ cells, genomic imprinting and dosage compensation to genome‐wide screens, evolution and human disease. These areas dovetailed well, converging ideas from various species and unifying seemingly divergent fields. Here, we highlight a few of the topics from an excellent programme of riveting news and novel findings. One of the main themes threading through this workshop was that of commitment, reprogramming and pluripotency. A. Surani (Cambridge, UK) discussed the initiation of epigenetic reprogramming in the germline. The main focus was on BLIMP1, which is an important regulator of germ cells that has roles in diverse processes, including the initiation of the PGC‐specification programme and exit from the pluripotent state. In this plenary address, Surani drew from several studies that illustrate the induction of cells to exit the pluripotent state and initiate the PGC‐specific programme, including the role of STELLA —a mammalian maternal‐effect gene that probably has a role in chromosomal organization. The BLIMP1 protein sets up chromatin signatures and influences the epigenetic reprogramming associated with X‐chromosome reactivation (Chuva de Sousa et al , 2008) and imprinting, emphasizing the links among pluripotency, reprogramming and commitment that were reiterated in several other talks. W. … [1]: /embed/graphic-1.gif

Drosophila development initiates with the formation of a diploid zygote followed by the rapid division of embryonic nuclei. This syncytial phase of development occurs almost entirely under maternal control and ends when the blastoderm embryo cellularizes and activates its zygotic genome. The biosynthesis and storage of histones in quantity sufficient for chromatin assembly of several thousands of genome copies represent a unique challenge for the developing embryo. In this article, we have reviewed our current understanding of the mechanisms involved in the production, storage, and deposition of histones in the fertilized egg and during the exponential amplification of cleavage nuclei.

In Drosophila, telomere-capping proteins have the remarkable capacity to recognize chromosome ends in a sequence-independent manner. This epigenetic protection is essential to prevent catastrophic ligations of chromosome extremities. Interestingly, capping proteins occupy a large telomere chromatin domain of several kilobases; however, the functional relevance of this to end protection is unknown. Here, we investigate the role of the large capping domain by manipulating HOAP (encoded by caravaggio) capping-protein expression in the male germ cells, where telomere protection can be challenged without compromising viability. We show that the exhaustion of HOAP results in a dramatic reduction of other capping proteins at telomeres, including K81 [encoded by ms(3)K81], which is essential for male fertility. Strikingly however, we demonstrate that, although capping complexes are barely detected in HOAP-depleted male germ cells, telomere protection and male fertility are not dramatically affected. Our study thus demonstrates that efficient protection of Drosophila telomeres can be achieved with surprisingly low amounts of capping complexes. We propose that these complexes prevent fusions by acting at the very extremity of chromosomes, reminiscent of the protection conferred by extremely short telomeric arrays in yeast or mammalian systems.

We have used in situ hybridization to analyze Na,K-ATPase alpha and beta subunit gene expression during zebrafish embryogenesis. The most striking finding is that each of the 14 Na,K-ATPase genes exhibits a distinct expression profile. All alpha and beta subunit genes are expressed in the nervous system, although the pattern of expression in different regions varies dramatically. In peripheral tissues, three of the five alpha1-like genes are expressed in pronephros and mucous cells, one is expressed in heart, and one is predominant in skeletal muscle. The alpha2 gene is expressed in brain and heart but is most prominent in skeletal muscle, while the two alpha3 genes are restricted in their expression to the nervous system. Of the six beta subunit genes, beta1a is expressed at highest abundance in lens, pronephros, and heart, while beta1b transcripts are abundant in mucous cells. The two beta2-like genes are differentially expressed in the nervous system. One beta3 gene is expressed exclusively in brain while the other is abundantly expressed in skeletal muscle. Based on these expression patterns, we predict that at least 14 alpha/beta subunit pairs are likely to be formed in different tissues.

We have identified cDNAs encoding a second zebrafish ortholog of the human Na,K-ATPase beta 2 subunit. The beta 2b cDNA encodes a 292 amino acid-long polypeptide with 74% identity to the previously characterized zebrafish beta 2a subunit. By using a zebrafish meiotic mapping panel, we determined that the beta 2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the beta 2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. beta 2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, beta 2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These results suggest that the beta 2a and beta 2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian beta 2 gene may be partitioned between the two zebrafish beta 2 orthologs.

The spermatozoon features an extremely condensed and inactive nucleus. The unique sperm chromatin organization is acquired during the late stages of spermatid differentiation by the replacement of somatic histones with sperm-specific chromosomal proteins. At fertilization, the inactive sperm nucleus must be rapidly transformed into a DNA replication competent male pronucleus before the formation of the zygote. The sequential events of this crucial process are well conserved among animals and are controlled by molecules present in the egg. We have previously identified a Drosophila maternal effect mutation called sésame, which specifically arrests male pronucleus formation at a late stage of chromatin decondensation. In this study, we show that sésame affects maternal histone incorporation in the male pronucleus, a situation that is expected to prevent nucleosomal organization of the paternal chromatin. As an apparent consequence, the male pronucleus is arrested before the first S-phase and does not condense mitotic chromosomes. However, centromeric heterochromatin is present on paternal centromeres, which occasionally interact with microtubules. The abnormal chromatin organization of the male pronucleus does not prevent the formation of a male pronuclear envelope, which breaks down and reassembles in synchrony with maternally derived nuclei present in the same cytoplasm.

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., Author Summary Chromosome organization relies on a basic functional unit called the nucleosome, in which DNA is wrapped around a core of histone proteins. However, during male gamete formation, the majority of histones are replaced by sperm-specific proteins that are adapted to sexual reproduction but incompatible with the formation of the first zygotic nucleus. These proteins must therefore be replaced by histones upon fertilization, in a replication-independent chromatin assembly process that requires the histone deposition factor HIRA. In this study, we identified the protein Yemanuclein (YEM) as a new partner of HIRA at fertilization. We show that, in eggs laid by yem mutant females, the male pronucleus fails to assemble its nucleosomes, resulting in the loss of paternal chromosomes at the first zygotic division. In addition, we found that YEM and HIRA are mutually dependent to perform chromatin assembly at fertilization, demonstrating that they tightly cooperate in vivo. Finally, we demonstrate that the replication-independent chromatin assembly factor ATRX/XNP is not involved in the assembly of paternal nucleosomes. In conclusion, our results shed new light into critical mechanisms controlling paternal chromosome formation at fertilization.

Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. Here, we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. We find that the Xnp chromatin remodeler and the Hira histone chaperone independently bind nucleosome-depleted chromatin. Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. We propose that Xnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus.

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

International audience; BACKGROUND: A critical function of telomeres is to prevent fusion of chromosome ends by the DNA repair machinery. In Drosophila somatic cells, assembly of the protecting capping complex at telomeres notably involves the recruitment of HOAP, HP1, and their recently identified partner, HipHop. We previously showed that the hiphop gene was duplicated before the radiation of the melanogaster subgroup of species, giving birth to K81, a unique paternal effect gene specifically expressed in the male germline. RESULTS: Here we show that K81 specifically associates with telomeres during spermiogenesis, along with HOAP and HP1, and is retained on paternal chromosomes until zygote formation. In K81 mutant testes, capping proteins are not maintained at telomeres in differentiating spermatids, resulting in the transmission of uncapped paternal chromosomes that fail to properly divide during the first zygotic mitosis. Despite the apparent similar capping roles of K81 and HipHop in their respective domain of expression, we demonstrate by in vivo reciprocal complementation analyses that they are not interchangeable. Strikingly, HipHop appeared to be unable to maintain capping proteins at telomeres during the global chromatin remodeling of spermatid nuclei. CONCLUSIONS: Our data demonstrate that K81 is essential for the maintenance of capping proteins at telomeres in postmeiotic male germ cells. In species of the melanogaster subgroup, HipHop and K81 have not only acquired complementary expression domains, they have also functionally diverged following the gene duplication event. We propose that K81 specialized in the maintenance of telomere protection in the highly peculiar chromatin environment of differentiating male gametes.

International audience; The nucleosomal organization of eukaryotic chromatin is generally established during DNA replication by the deposition of canonical histones synthesized in S phase. However, cells also use a Replication Independent (RI) nucleosome assembly pathway that allows the incorporation of non-canonical histone variants in the chromatin. H3.3 is a conserved histone variant that is structurally very close to its canonical counterpart but nevertheless possesses specific properties. In this review, we discuss the dual role of H3.3 which functions as a neutral replacement histone, but also participates in the epigenetic transmission of active chromatin states. These properties of H3.3 are also explored in the light of recent studies that implicate this histone and its associated chromatin assembly factors in large scale, replication-independent chromatin remodeling events. In particular, H3.3 appears as a critical player in the transmission of the paternal genome, from sperm to zygote.

Wolbachia is a bacteria endosymbiont that rapidly infects insect populations through a mechanism known as cytoplasmic incompatibility (CI). In CI, crosses between Wolbachia-infected males and uninfected females produce severe cell cycle defects in the male pronucleus resulting in early embryonic lethality. In contrast, viable progeny are produced when both parents are infected (the Rescue cross). An important consequence of CI–Rescue is that infected females have a selective advantage over uninfected females facilitating the rapid spread of Wolbachia through insect populations. CI disrupts a number of prophase and metaphase events in the male pronucleus, including Cdk1 activation, chromosome condensation, and segregation. Here, we demonstrate that CI disrupts earlier interphase cell cycle events. Specifically, CI delays the H3.3 and H4 deposition that occurs immediately after protamine removal from the male pronucleus. In addition, we find prolonged retention of the replication factor PCNA in the male pronucleus into metaphase, indicating progression into mitosis with incompletely replicated DNA. We propose that these CI-induced interphase defects in de novo nucleosome assembly and replication are the cause of the observed mitotic condensation and segregation defects. In addition, these interphase chromosome defects likely activate S-phase checkpoints, accounting for the previously described delays in Cdk1 activation. These results have important implications for the mechanism of Rescue and other Wolbachia-induced phenotypes., Author Summary Wolbachia are among the most successful of all intracellular bacteria, infecting an estimated 65% of insect species. Wolbachia are also present in filarial nematodes and are the cause of African river blindness. Wolbachia's success is due in part to its ability to induce a conditional form of sterility known as cytoplasmic incompatibility (CI), endowing infected females with a tremendous selective advantage. CI results in the severe reduction in progeny from crosses between uninfected females and Wolbachia-infected males. However, Wolbachia-infected females can mate with either infected or uninfected males with no reduction in progeny. CI may drive speciation and is intensively being pursued as a means to control insect-borne human disease. In spite of its biological and medical significance, the molecular basis of CI is not understood. We take advantage of newly generated chromatin reagents to demonstrate that prior to the well-documented defects in chromosome condensation and segregation, CI produces a delay in recruiting the replication-independent histone H3.3/H4 complex to the male pronucleus. There is great interest in histone H3.3 because of its general role in transcription and in remodeling of the sperm chromatin following fertilization. In addition, these findings may provide insight into other Wolbachia–host interactions such as CI–Rescue and male-killing.