Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphology but are dispensable for male fertility.
01 Nov 2014-G3: Genes, Genomes, Genetics (Genetics Society of America)-Vol. 4, Iss: 11, pp 2241-2245
TL;DR: Drosophila males homozygous for a genomic deletion covering several genes including the protamine-like genes Mst35Ba/b are surprisingly fertile, and this work precisely deleted the Mst 35B locus by homologous recombination, and it is confirmed the dispensability of Mst34B for fertility.
Abstract: 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.
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TL;DR: CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets and this is proposed to be a rescue mechanism for embryos.
Abstract: Intracellular Wolbachia bacteria manipulate arthropod reproduction to promote their own inheritance. The most prevalent mechanism, cytoplasmic incompatibility (CI), traces to a Wolbachia deubiquitylase, CidB, and CidA. CidB has properties of a toxin, while CidA binds CidB and rescues embryonic viability. CidB is also toxic to yeast where we identified both host effects and high-copy suppressors of toxicity. The strongest suppressor was karyopherin-α, a nuclear-import receptor; this required nuclear localization-signal binding. A protein-interaction screen of Drosophila extracts using a substrate-trapping catalytic mutant, CidB*, also identified karyopherin-α; the P32 protamine-histone exchange factor bound as well. When CidB* bound CidA, these host protein interactions disappeared. These associations would place CidB at the zygotic male pronucleus where CI defects first manifest. Overexpression of karyopherin-α, P32, or CidA in female flies suppressed CI. We propose that CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets.
63 citations
Cites background from "Drosophila protamine-like Mst35Ba a..."
...PCR amplicons were produced with primers listed in Supplementary file 1i. High fidel- ity Phusion polymerase (New England Biolabs) was used to amplify DNA, which was then restriction enzyme digested, gel-purified and ligated into various plasmid vectors (Supplementary file 1j)....
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...In order to precipitate and remove DNA, we added 5 M NaCl while stirring to a final concentration of 1 M and poly(ethyleneimine) (PEI) from a stock of 10% PEI in 10% HCl to a final concentration ~0.3–0.5%....
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...The small, highly basic protamine proteins used to package paternal DNA at high density are stripped from the DNA, (Balhorn, 2007; Rathke et al., 2014; Tirmarche et al., 2014; Loppin et al., 2015; Tirmarche et al., 2016) and nucleosomes are then assembled with maternal histones (Loppin et al., 2015; Liu et al., 1997)....
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...The small, highly basic protamine proteins used to package paternal DNA at high density are stripped from the DNA, (Balhorn, 2007; Rathke et al., 2014; Tirmarche et al., 2014; Loppin et al., 2015; Tirmarche et al., 2016) and nucleosomes are then assembled with maternal histones (Loppin et al....
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TL;DR: This review synthesize and summarize the current knowledge on the progress of chromatin remodeling during spermiogenesis, and straighten out the chronological order of chromatis remodeling and illustrate the possible regulation mechanisms of each step.
51 citations
TL;DR: The current knowledge of fertilization in Drosophila melanogaster is reviewed, with a special emphasis on the genes involved in the complex transformation of the fertilizing sperm nucleus into a replicated set of paternal chromosomes.
Abstract: 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 cellul...
45 citations
TL;DR: It is demonstrated that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization and is then rapidly degraded after fertilization.
Abstract: 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.
37 citations
TL;DR: It is demonstrated that Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus and leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links.
Abstract: 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.
21 citations
References
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TL;DR: In this article, the sperm DNA is packaged very tightly to protect the DNA during the transit that occurs before fertilization, however, this condensation cannot sacrifice chromosomal elements that are essential for the embryo to access the correct sequences of the paternal genome for proper initiation of the embryonic developmental program.
Abstract: Understanding how DNA is packaged in the mammalian sperm cell has important implications for human infertility as well as for the cell biology. Recent advances in the study of mammalian sperm chromatin structure and function have altered our perception of this highly condensed, inert chromatin. Sperm DNA is packaged very tightly to protect the DNA during the transit that occurs before fertilization. However, this condensation cannot sacrifice chromosomal elements that are essential for the embryo to access the correct sequences of the paternal genome for proper initiation of the embryonic developmental program. The primary levels of the sperm chromatin structure can be divided into three main categories: the large majority of DNA is packaged by protamines, a smaller amount (2-15%) retains histone-bound chromatin and the DNA is attached to the nuclear matrix at roughly 50 kb intervals. Current data suggest that the latter two structural elements are transferred to the paternal pronucleus after fertilization where they have important functional roles. The nuclear matrix organization is essential for DNA replication, and the histone-bound chromatin identifies genes that are important for embryonic development. These data support the emerging view of the sperm genome as providing, in addition to the paternal DNA sequence, a structural framework that includes molecular regulatory factors that are required for proper embryonic development.
314 citations
TL;DR: This work focuses on post-meiotic stages and shows that also after meiosis, histone H3 shows a high overall methylation of K9 and K27 and hypothesise that these modifications ensure maintenance of transcriptional silencing in the haploid genome.
Abstract: In higher organisms, the chromatin of sperm is organised in a highly condensed protamine-based structure. In pre-meiotic stages and shortly after meiosis, histones carry multiple modifications. Here, we focus on post-meiotic stages and show that also after meiosis, histone H3 shows a high overall methylation of K9 and K27 and we hypothesise that these modifications ensure maintenance of transcriptional silencing in the haploid genome. Furthermore, we show that histones are lost during the early canoe stage and that just before this stage, hyper-acetylation of histone H4 and mono-ubiquitylation of histone H2A occurs. We believe that these histone modifications within the histone-based chromatin architecture may lead to better access of enzymes and chromatin remodellers. This notion is supported by the presence of the architectural protein CTCF, numerous DNA breaks, SUMO, UbcD6 and high content of ubiquitin, as well as testes-specific nuclear proteasomes at this time. Moreover, we report the first transition protein-like chromosomal protein, Tpl(94D), to be found in Drosophila. We propose that Tpl(94D)--an HMG box protein--and the numerous DNA breaks facilitate chromatin unwinding as a prelude to protamine and Mst77F deposition. Finally, we show that histone modifications and removal are independent of protamine synthesis.
201 citations
"Drosophila protamine-like Mst35Ba a..." refers background or methods in this paper
...Finally, using a specific antibody (Figure S2), we also verified that the transition protein Tpl94D (Rathke et al. 2007) was normally incorporated in mutant spermatids at the histone-to-protamine transition (Figure 2, D and E), confirming that the nuclear defects in mutant spermatids appear after this stage....
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...Volume 4 November 2014 | Function of Protamine-Like Proteins | 2243...
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...Finally, using a specific antibody (Figure S2), we also verified that the transition protein Tpl94D (Rathke et al. 2007) was normally incorporated in mutant spermatids at the histone-to-protamine transition (Figure 2, D and E), confirming that the nuclear defects in mutant spermatids appear after…...
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TL;DR: HILS1 displays several biochemical properties that are similar to those of linker histones, including the abilities to bind reconstituted mononucleosomes, produce a chromatosome stop during micrococcal nuclease digestion, and aggregate chromatin.
Abstract: Chromatin remodeling is a major event that occurs during mammalian spermiogenesis, the process of spermatid maturation into spermatozoa. Nuclear condensation during spermiogenesis is accomplished by replacing somatic histones (linker histone H1 and core histones) and the testis-specific linker histone, H1t, with transition proteins and protamines. It has long been thought that H1t is the only testis-specific linker histone, and that all linker histones are replaced by transition proteins, and subsequently by protamines during spermiogenesis. Here, we report the identification and characterization of a spermatid-specific linker histone H1-like protein (termed HILS1) in the mouse and human. Both mouse and human HILS1 genes are located in intron 8 of the α-sarcoglycan genes. HILS1 is highly expressed in nuclei of elongating and elongated spermatids (steps 9-15). HILS1 displays several biochemical properties that are similar to those of linker histones, including the abilities to bind reconstituted mononucleosomes, produce a chromatosome stop during micrococcal nuclease digestion, and aggregate chromatin. Because HILS1 is expressed in late spermatids that do not contain core histones, HILS1 may participate in spermatid nuclear condensation through a mechanism distinct from that of linker histones. Because HILS1 also belongs to the large winged helix/forkhead protein superfamily, HILS1 may also regulate gene transcription, DNA repair, and/or other chromosome processes during mammalian spermiogenesis.
180 citations
"Drosophila protamine-like Mst35Ba a..." refers background in this paper
...Mst77F, which was originally identified in a genetic screen for b2 tubulin interactors (Fuller et al. 1989), is related to the mammalian spermatid-specific histone H1-like protein HILS1 (Iguchi et al. 2004; Yan et al. 2003)....
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TL;DR: An updated comparative analysis of protamines and their corresponding genes is presented, including representative organisms from each of the vertebrate classes and one invertebrate (squid, Loligo opalescens).
Abstract: An updated comparative analysis of protamines and their corresponding genes is presented, including representative organisms from each of the vertebrate classes and one invertebrate (squid, Loligo opalescens). Special emphasis is placed on the implications for sperm chromatin organization and the evolutionary significance. The review is based on some of the most recent publications in the field and builds upon previously published reviews on this topic.
174 citations
"Drosophila protamine-like Mst35Ba a..." refers background in this paper
...Miller, D., M. Brinkworth, and D. Iles, 2010 Paternal DNA packaging in spermatozoa: more than the sum of its parts?...
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TL;DR: The data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals, including protamines and linker histone-like protein.
Abstract: Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little is known for Drosophila melanogaster. Here we characterize three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame.
156 citations
"Drosophila protamine-like Mst35Ba a..." refers background in this paper
...Rathke, C., B. Barckmann, S. Burkhard, S. Jayaramaiah-Raja, J. Roote et al., 2010 Distinct functions of Mst77F and protamines in nuclear shaping and chromatin condensation during Drosophila spermiogenesis....
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...Jayaramaiah Raja, S., and R. Renkawitz-Pohl, 2005 Replacement by Drosophila melanogaster protamines and Mst77F of histones during chromatin condensation in late spermatids....
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...Mst35Ba/b proteins are incorporated in elongating spermatid nuclei at the late canoe stage and remain associated with mature sperm nuclei until fertilization (Jayaramaiah Raja and RenkawitzPohl 2005)....
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...Volume 4 November 2014 | Function of Protamine-Like Proteins | 2243...
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...Drosophila comprises at least three SNBPs: two paralogous protamine-like proteins, Mst35Ba and Mst35Bb, which are conserved among drosophilids, and the HILS1related protein Mst77F (Russell and Kaiser 1993; Jayaramaiah Raja and Renkawitz-Pohl 2005; Alvi et al. 2013; Rathke et al. 2014)....
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