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1.
Nat Commun ; 14(1): 4187, 2023 07 13.
Article in English | MEDLINE | ID: mdl-37443316

ABSTRACT

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.


Subject(s)
Gryllidae , Animals , Male , Gryllidae/genetics , Semen/metabolism , Chromatin/genetics , Chromatin/metabolism , Spermatogenesis/genetics , Histones/metabolism , Spermatozoa/metabolism
2.
PLoS Genet ; 18(1): e1009615, 2022 01.
Article in English | MEDLINE | ID: mdl-34982772

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/physiology , Heterochromatin/genetics , Histone Demethylases/metabolism , Membrane Proteins/genetics , RNA-Binding Proteins/metabolism , Sin3 Histone Deacetylase and Corepressor Complex/metabolism , Thioredoxins/genetics , Transcription Factors/metabolism , Animals , Epigenomics , Female , Gene Expression Regulation , Heterochromatin/chemistry , Histones/metabolism , Male , Maternal Inheritance , Organ Specificity , Ovary/chemistry , Promoter Regions, Genetic , Regulatory Elements, Transcriptional
3.
Nat Struct Mol Biol ; 27(11): 1057-1068, 2020 11.
Article in English | MEDLINE | ID: mdl-32895554

ABSTRACT

Nucleosomes represent a challenge in regard to transcription. Histone eviction enables RNA polymerase II (RNAPII) progression through DNA, but compromises chromatin integrity. Here, we used the SNAP-tag system to distinguish new and old histones and monitor chromatin reassembly coupled to transcription in human cells. We uncovered a transcription-dependent loss of old histone variants H3.1 and H3.3. At transcriptionally active domains, H3.3 enrichment reflected both old H3.3 retention and new deposition. Mechanistically, we found that the histone regulator A (HIRA) chaperone is critical to processing both new and old H3.3 via different pathways. De novo H3.3 deposition is totally dependent on HIRA trimerization as well as on its partner ubinuclein 1 (UBN1), while antisilencing function 1 (ASF1) interaction with HIRA can be bypassed. By contrast, recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows absolute dependency on ASF1-HIRA interaction. We propose a model whereby HIRA coordinates these distinct pathways during transcription to fine-tune chromatin states.


Subject(s)
Cell Cycle Proteins/metabolism , Histone Chaperones/metabolism , Histones/metabolism , Signal Transduction , Transcription Factors/metabolism , Transcription, Genetic , HeLa Cells , Histones/genetics , Humans , Nuclear Proteins/metabolism , Protein Multimerization
4.
PLoS Genet ; 16(3): e1008543, 2020 03.
Article in English | MEDLINE | ID: mdl-32134927

ABSTRACT

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.


Subject(s)
Drosophila Proteins/genetics , Histone Demethylases/genetics , Oocytes/physiology , Zygote/physiology , Animals , Cell Nucleus/genetics , Chromatin/genetics , Chromatin Assembly and Disassembly/genetics , Drosophila/genetics , Female , Gene Expression Regulation, Developmental/genetics , Histones/genetics , Male , Oogenesis/genetics , Spermatozoa/physiology , Transcription, Genetic/genetics
5.
Nat Commun ; 9(1): 3181, 2018 08 09.
Article in English | MEDLINE | ID: mdl-30093638

ABSTRACT

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated. Replication stress further complicates the safeguard of epigenome integrity. Here, we investigate the transmission of the histone variants H3.3 and H3.1 during replication. We follow their distribution relative to replication timing, first in the genome and, second, in 3D using super-resolution microscopy. We find that H3.3 and H3.1 mark early- and late-replicating chromatin, respectively. In the nucleus, H3.3 forms domains, which decrease in density throughout replication, while H3.1 domains increase in density. Hydroxyurea impairs local recycling of parental histones at replication sites. Similarly, depleting the histone chaperone ASF1 affects recycling, leading to an impaired histone variant landscape. We discuss how faithful transmission of histone variants involves ASF1 and can be impacted by replication stress, with ensuing consequences for cell fate and tumorigenesis.


Subject(s)
Cell Cycle Proteins/chemistry , Chromatin/chemistry , DNA Replication , Histones/chemistry , Cell Lineage , DNA/chemistry , Epigenesis, Genetic , Genome, Human , HeLa Cells , Humans , Hydroxyurea/chemistry , Microscopy , Microscopy, Fluorescence , Molecular Chaperones , Nucleosomes/chemistry , S Phase
6.
Methods Mol Biol ; 1832: 207-221, 2018.
Article in English | MEDLINE | ID: mdl-30073529

ABSTRACT

Distinct histone variants mark chromatin domains in the nucleus. To understand how these marks are established and maintained, one has to decipher how the dynamic distribution of these variants is orchestrated. These dynamics are associated with all DNA-based processes such as DNA replication, repair, transcription, heterochromatin formation and chromosome segregation. Key factors, known as histone chaperones, have been involved in escorting histones, thereby contributing to the chromatin landscape of given cell types. SNAP-tag-based imaging system enables the distinction between old and newly deposited histones, and has proved to be a powerful method for the visualization of histone variant dynamics on a cell-by-cell basis. This approach enables the tracking of specific variants in vivo and defining their timing and mode of deposition throughout the cell cycle and in different nuclear territories. Here, we provide a detailed protocol to exploit the SNAP-tag technology to assess the dynamics of newly synthesized and old histones. We then show that combining the SNAP-tagging of histones with the knockdown of candidate factors, represents an effective approach to decipher the role of key actors in guiding histone dynamics. Here, we specifically illustrate how this strategy was used to identify the essential role of the chaperone HIRA in deposition of newly synthesized histone variant H3.3.


Subject(s)
Histone Chaperones/metabolism , Histones/biosynthesis , Imaging, Three-Dimensional , Molecular Biology/methods , HeLa Cells , Humans , Protein Isoforms/biosynthesis , Staining and Labeling
7.
Genes Dev ; 31(5): 463-480, 2017 03 01.
Article in English | MEDLINE | ID: mdl-28356341

ABSTRACT

In mammals, centromere definition involves the histone variant CENP-A (centromere protein A), deposited by its chaperone, HJURP (Holliday junction recognition protein). Alterations in this process impair chromosome segregation and genome stability, which are also compromised by p53 inactivation in cancer. Here we found that CENP-A and HJURP are transcriptionally up-regulated in p53-null human tumors. Using an established mouse embryonic fibroblast (MEF) model combining p53 inactivation with E1A or HRas-V12 oncogene expression, we reproduced a similar up-regulation of HJURP and CENP-A. We delineate functional CDE/CHR motifs within the Hjurp and Cenpa promoters and demonstrate their roles in p53-mediated repression. To assess the importance of HJURP up-regulation in transformed murine and human cells, we used a CRISPR/Cas9 approach. Remarkably, depletion of HJURP leads to distinct outcomes depending on their p53 status. Functional p53 elicits a cell cycle arrest response, whereas, in p53-null transformed cells, the absence of arrest enables the loss of HJURP to induce severe aneuploidy and, ultimately, apoptotic cell death. We thus tested the impact of HJURP depletion in pre-established allograft tumors in mice and revealed a major block of tumor progression in vivo. We discuss a model in which an "epigenetic addiction" to the HJURP chaperone represents an Achilles' heel in p53-deficient transformed cells.


Subject(s)
Autoantigens/metabolism , Cell Transformation, Neoplastic/genetics , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Genes, p53/genetics , Oncogenes/genetics , Amino Acid Motifs/genetics , Animals , Autoantigens/genetics , Cell Line , Cells, Cultured , Centromere Protein A , Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation/genetics , DNA-Binding Proteins/genetics , Female , Gene Deletion , Genomic Instability/genetics , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Models, Animal
8.
Cell Stem Cell ; 19(5): 567-568, 2016 11 03.
Article in English | MEDLINE | ID: mdl-27814477

ABSTRACT

The molecular features underlying tumor heterogeneity and the role of chromatin components in regulating cell fate within tumors are not well understood. Recently in Science, Torres et al. (2016) showed that the linker histone variant H1.0 functions as a chromatin switch that determines self-renewal versus differentiation decisions in cancer stem cells.


Subject(s)
Chromatin , Histones/genetics , Cell Differentiation , Humans , Neoplastic Stem Cells
9.
Curr Protoc Mol Biol ; 110: 21.31.1-21.31.25, 2015 Apr 01.
Article in English | MEDLINE | ID: mdl-25827087

ABSTRACT

Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin (ORGANIC) is a high-resolution method that can be used to quantitatively map protein-DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low-salt solubilization to preserve protein-DNA complexes, followed by immunoprecipitation and paired-end sequencing for genome-wide mapping of binding sites. In this unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein-DNA complexes, and high-throughput sequencing to map sites of bound factors.


Subject(s)
Chromatin Immunoprecipitation/methods , Chromatin/genetics , Chromatin/metabolism , Transcription Factors/metabolism , Binding Sites , Chromatin/chemistry , High-Throughput Nucleotide Sequencing , Macromolecular Substances/isolation & purification , Protein Binding , Saccharomyces cerevisiae/genetics
10.
Curr Biol ; 24(19): 2281-7, 2014 Oct 06.
Article in English | MEDLINE | ID: mdl-25242033

ABSTRACT

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.


Subject(s)
Chromosomes/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Animals , DNA Replication , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Haploidy , Zygote/metabolism
11.
Genome Res ; 24(5): 809-20, 2014 May.
Article in English | MEDLINE | ID: mdl-24668908

ABSTRACT

Polycomb-mediated chromatin repression modulates gene expression during development in metazoans. Binding of multiple sequence-specific factors at discrete Polycomb response elements (PREs) is thought to recruit repressive complexes that spread across an extended chromatin domain. To dissect the structure of PREs, we applied high-resolution mapping of nonhistone chromatin proteins in native chromatin of Drosophila cells. Analysis of occupied sites reveal interactions between transcription factors that stabilize Polycomb anchoring to DNA, and implicate the general transcription factor ADF1 as a novel PRE component. By comparing two Drosophila cell lines with differential chromatin states, we provide evidence that repression is accomplished by enhanced Polycomb recruitment both to PREs and to target promoters of repressed genes. These results suggest that the stability of multifactor complexes at promoters and regulatory elements is a crucial aspect of developmentally regulated gene expression.


Subject(s)
Chromatin Assembly and Disassembly , Drosophila/genetics , Polycomb-Group Proteins/metabolism , Response Elements/genetics , Animals , Cells, Cultured , Drosophila/metabolism , Drosophila Proteins/metabolism , Protein Binding , Transcription Factors/metabolism
12.
Nat Methods ; 11(2): 203-9, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24336359

ABSTRACT

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. TF profiling is commonly carried out by formaldehyde cross-linking and sonication followed by chromatin immunoprecipitation (X-ChIP). We describe a method to profile TF binding at high resolution without cross-linking. We begin with micrococcal nuclease-digested non-cross-linked chromatin and then perform affinity purification of TFs and paired-end sequencing. The resulting occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide high-resolution maps that are accurate, as defined by the presence of known TF consensus motifs in identified binding sites, that are not biased toward accessible chromatin and that do not require input normalization. We profiled Drosophila melanogaster GAGA factor and Pipsqueak to test ORGANIC performance on larger genomes. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.


Subject(s)
Chromatin Immunoprecipitation/methods , Chromatin/metabolism , Computational Biology , Drosophila melanogaster/metabolism , Genome, Fungal , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Animals , Binding Sites , Chromatin/genetics , DNA Footprinting , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/genetics
13.
PLoS Genet ; 9(2): e1003285, 2013.
Article in English | MEDLINE | ID: mdl-23408912

ABSTRACT

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.


Subject(s)
Cell Cycle Proteins , DNA-Binding Proteins , Drosophila Proteins , Histone Chaperones , Nuclear Proteins , Nucleosomes , Transcription Factors , Zygote , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromatin/metabolism , Chromatin/ultrastructure , Chromatin Assembly and Disassembly , DNA Replication/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Fertilization/genetics , Histone Chaperones/genetics , Histone Chaperones/metabolism , Histones/genetics , Histones/metabolism , Male , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleosomes/genetics , Nucleosomes/metabolism , Spermatozoa/cytology , Spermatozoa/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Zygote/growth & development , Zygote/metabolism
14.
Proc Natl Acad Sci U S A ; 109(48): 19721-6, 2012 Nov 27.
Article in English | MEDLINE | ID: mdl-23150573

ABSTRACT

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.


Subject(s)
Chromatin/metabolism , Histones/metabolism , Nucleosomes/metabolism , Animals , Chromatin Immunoprecipitation , Drosophila , HSP70 Heat-Shock Proteins/genetics
15.
PLoS Biol ; 10(12): e1001434, 2012.
Article in English | MEDLINE | ID: mdl-23300376

ABSTRACT

In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric 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.


Subject(s)
Centromere/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Histones/metabolism , Inheritance Patterns/genetics , Spermatozoa/metabolism , Animals , Centromere Protein A , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/metabolism , Drosophila melanogaster/embryology , Embryonic Development/genetics , Fertilization , G2 Phase/genetics , Green Fluorescent Proteins/metabolism , Kinetochores/metabolism , Male , Recombinant Fusion Proteins/metabolism , Spermatogenesis/genetics
16.
Curr Biol ; 20(23): 2090-9, 2010 Dec 07.
Article in English | MEDLINE | ID: mdl-21093267

ABSTRACT

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.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Spermatozoa/physiology , Telomere/metabolism , Animals , Animals, Genetically Modified , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Drosophila Proteins/genetics , Epigenesis, Genetic , Female , Male , Multigene Family , Phylogeny , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
17.
J Cell Sci ; 123(Pt 20): 3515-24, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-20841382

ABSTRACT

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.


Subject(s)
Chimera/genetics , Drosophila/genetics , Drosophila/physiology , Infertility/genetics , Retroelements/genetics , Animals , Cell Nucleus/genetics , Cell Nucleus/metabolism , DNA Damage/genetics , Female , Haploidy , Male , Meiosis , Oocytes/cytology , Oocytes/metabolism , Zygote
18.
Int J Dev Biol ; 53(2-3): 231-43, 2009.
Article in English | MEDLINE | ID: mdl-19412883

ABSTRACT

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.


Subject(s)
Embryo, Nonmammalian/metabolism , Epigenesis, Genetic , Histones/metabolism , Amino Acid Sequence , Animals , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/embryology , Genetic Variation , Histones/genetics , Histones/physiology , Models, Biological , Molecular Sequence Data , Reproduction/genetics , Reproduction/physiology , Sequence Homology, Amino Acid
19.
PLoS Pathog ; 5(3): e1000343, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19300496

ABSTRACT

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.


Subject(s)
Drosophila/microbiology , Histones/metabolism , Host-Pathogen Interactions/genetics , Rickettsia Infections/genetics , Wolbachia/physiology , Animals , Animals, Genetically Modified , Cell Nucleus , Drosophila/genetics , Embryo, Nonmammalian , Female , Fluorescent Antibody Technique , Infertility, Male/genetics , Infertility, Male/microbiology , Male , Microscopy, Confocal , Spermatozoa/pathology
20.
PLoS Genet ; 3(10): 1991-2006, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17967064

ABSTRACT

In many animal species, the sperm DNA is packaged with male germ line--specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male pronucleus. By studying a point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male pronucleus. We report here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3 deposition is apparently not affected in Hira mutant embryos and adults, suggesting that different chromatin assembly machineries could deposit this histone variant.


Subject(s)
Cell Cycle Proteins/metabolism , Chromatin Assembly and Disassembly , Chromatin/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Fertilization/physiology , Transcription Factors/metabolism , Alleles , Animals , Cell Cycle Proteins/genetics , Cell Nucleus/metabolism , DNA Replication , Drosophila Proteins/genetics , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Fathers , Female , Gene Targeting , Genes, Insect , Histone Chaperones , Histones/metabolism , Male , Molecular Chaperones/metabolism , Mutant Proteins/metabolism , Mutation/genetics , Ovum/cytology , Ovum/metabolism , Phenotype , Protamines/isolation & purification , Recombination, Genetic/genetics , Spermatozoa/cytology , Spermatozoa/metabolism , Transcription Factors/genetics
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