Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphology but are dispensable for male fertility.

doi:10.1534/G3.114.012724

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Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphology but are dispensable for male fertility.

Journal Article•DOI•

Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphology but are dispensable for male fertility.

Samantha Tirmarche¹, Shuhei Kimura¹, Laure Sapey-Triomphe¹, William J. Sullivan², Frédéric Landmann³, Benjamin Loppin¹ - Show less +2 more•Institutions (3)

Claude Bernard University Lyon 1¹, University of California, Santa Cruz², Centre national de la recherche scientifique³

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.

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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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Journal Article•DOI•

The Wolbachia cytoplasmic incompatibility enzyme CidB targets nuclear import and protamine-histone exchange factors

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John F. Beckmann¹, Gagan Deep Sharma¹, Luis Mendez¹, Hongli Chen², Mark Hochstrasser² - Show less +1 more•Institutions (2)

Auburn University¹, Yale University²

27 Nov 2019-eLife

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.

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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.

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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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Journal Article•DOI•

The dynamics and regulation of chromatin remodeling during spermiogenesis.

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Shuang-Li Hao¹, Fei-Da Ni¹, Wan-Xi Yang¹•Institutions (1)

Zhejiang University¹

20 Jul 2019-Gene

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.

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51 citations

Journal Article•DOI•

The intimate genetics of Drosophila fertilization

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Benjamin Loppin¹, Raphaëlle Dubruille¹, Béatrice Horard¹•Institutions (1)

Claude Bernard University Lyon 1¹

01 Aug 2015-Open Biology

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.

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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...

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45 citations

Journal Article•DOI•

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

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Samantha Tirmarche¹, Shuhei Kimura¹, Raphaëlle Dubruille¹, Béatrice Horard¹, Benjamin Loppin¹ - Show less +1 more•Institutions (1)

University of Lyon¹

23 Nov 2016-Nature Communications

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.

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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.

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37 citations

Journal Article•DOI•

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

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Shuhei Kimura¹, Benjamin Loppin¹•Institutions (1)

University of Lyon¹

01 Nov 2016-Open Biology

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.

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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.

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21 citations

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Journal Article•DOI•

The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization.

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Emilie Bonnefoy¹, Guillermo A. Orsi¹, Guillermo A. Orsi², Pierre Couble¹, Pierre Couble², Benjamin Loppin², Benjamin Loppin¹ - Show less +3 more•Institutions (2)

University of Lyon¹, Centre national de la recherche scientifique²

01 Jan 2005-PLOS Genetics

TL;DR: Analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination revealed that the only essential function of HIRA is the assembly of paternal chromatin during male pronucleus formation, and established that protamine removal and histone deposition are two functionally distinct processes.

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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.

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129 citations

Journal Article•DOI•

Genomic Deletions of the Drosophila melanogaster Hsp70 Genes

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Wei J. Gong¹, Kent G. Golic¹•Institutions (1)

University of Utah¹

01 Nov 2004-Genetics

TL;DR: The results show that genomic deletions of varied sizes can be readily generated by homologous recombination in Drosophila, including one deletion that spanned ∼47 kb.

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Abstract: Homologous recombination can produce directed mutations in the genomes of a number of model organisms, including Drosophila melanogaster. One of the most useful applications has been to delete target genes to generate null alleles. In Drosophila, specific gene deletions have not yet been produced by this method. To test whether such deletions could be produced by homologous recombination in D. melanogaster we set out to delete the Hsp70 genes. Six nearly identical copies of this gene, encoding the major heat-shock protein in Drosophila, are found at two separate but closely linked loci. This arrangement has thwarted standard genetic approaches to generate an Hsp70-null fly, making this an ideal test of gene targeting. In this study, ends-out targeting was used to generate specific deletions of all Hsp70 genes, including one deletion that spanned ∼47 kb. The Hsp70-null flies are viable and fertile. The results show that genomic deletions of varied sizes can be readily generated by homologous recombination in Drosophila.

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115 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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...Yan, W., L. Ma, K. H. Burns, and M. M. Matzuk, 2003 HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis....
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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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Journal Article•DOI•

Distinct functions of Mst77F and protamines in nuclear shaping and chromatin condensation during Drosophila spermiogenesis

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Christina Rathke¹, Bridlin Barckmann¹, Silja Burkhard¹, Sunil Jayaramaiah-Raja¹, John Roote², Renate Renkawitz-Pohl¹ - Show less +2 more•Institutions (2)

University of Marburg¹, University of Cambridge²

01 Apr 2010-European Journal of Cell Biology

TL;DR: Data support the long-standing hypothesis that the switch from a histone- to protamine-based chromatin protects the paternal genome from mutagens.

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86 citations

"Drosophila protamine-like Mst35Ba a..." refers background or result in this paper

...It has been proposed that Mst77F is essential for male fertility (Rathke et al. 2010), but this conclusion is based on the analysis of the antimorphic point mutation Mst77F1 (see Krsticevic et al. 2010)....
[...]
...Although the functions of Drosophila SNBPs remain poorly understood, Rathke et al. (2010) reported the surprising observation that Drosophila males homozygous for a genomic deficiency covering the Mst35B locus were fertile....
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...Although the quantity and quality of gametes were affected by the loss of Mst35B genes, homozygous DMst35B and DMst35B/ protD males were nevertheless fertile, in agreement with the study by Rathke et al. (2010)....
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...It is likely that the concentration of chromatin at one end of mutant spermatid nuclei observed by Rathke et al. (2010) actually correspond to folded nuclear extremities....
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...Volume 4 November 2014 | Function of Protamine-Like Proteins | 2243...
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Journal Article•DOI•

Functional Copies of the Mst77F Gene on the Y Chromosome of Drosophila melanogaster

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Flavia Krsticevic¹, Henrique L. Santos¹, Suelen Januário¹, Carlos G. Schrago¹, A. Bernardo Carvalho¹ - Show less +1 more•Institutions (1)

Federal University of Rio de Janeiro¹

01 Jan 2010-Genetics

TL;DR: The identification of functional Mst77Y genes reinforces the previous finding that gene gains play a prominent role in the evolution of the Drosophila Y chromosome.

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Abstract: The Y chromosome of Drosophila melanogaster has <20 protein-coding genes. These genes originated from the duplication of autosomal genes and have male-related functions. In 1993, Russell and Kaiser found three Y-linked pseudogenes of the Mst77F gene, which is a testis-expressed autosomal gene that is essential for male fertility. We did a thorough search using experimental and computational methods and found 18 Y-linked copies of this gene (named Mst77Y-1-Mst77Y-18). Ten Mst77Y genes encode defective proteins and the other eight are potentially functional. These eight genes produce approximately 20% of the functional Mst77F-like mRNA, and molecular evolutionary analysis shows that they evolved under purifying selection. Hence several Mst77Y genes have all the features of functional genes. Mst77Y genes are present only in D. melanogaster, and phylogenetic analysis confirmed that the duplication is a recent event. The identification of functional Mst77Y genes reinforces the previous finding that gene gains play a prominent role in the evolution of the Drosophila Y chromosome.

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41 citations

"Drosophila protamine-like Mst35Ba a..." refers background in this paper

...Interestingly, the D. melanogaster genome contains several recent copies of Mst77F on the Y chromosome, and eight of these Mst77Y genes are most likely functional (Russell and Kaiser 1993; Krsticevic et al. 2010)....
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...It has been proposed that Mst77F is essential for male fertility (Rathke et al. 2010), but this conclusion is based on the analysis of the antimorphic point mutation Mst77F1 (see Krsticevic et al. 2010)....
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Journal Article•DOI•

Drosophila melanogaster male germ line-specific transcripts with autosomal and Y-linked genes.

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Steven Russell¹, Kim Kaiser¹•Institutions (1)

University of Glasgow¹

01 May 1993-Genetics

TL;DR: A set of related transcripts expressed in the germ line of male Drosophila melanogaster are identified, while one of the corresponding genes is autosomal the remainder are located on the Y chromosome, suggesting that the D. melanogasters Y chromosome contains functional protein-coding genes.

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Abstract: We have identified of set of related transcripts expressed in the germ line of male Drosophila melanogaster. Surprisingly, while one of the corresponding genes is autosomal the remainder are located on the Y chromosome. The autosomal locus, at 77F on chromosome arm 3L, corresponds to the previously described transcription unit 18c, located in the first intron of the gene for an RI subunit of cAMP-dependent protein kinase. The Y chromosome copies have been mapped to region h18-h19 on the cytogenetic map of the Y outside of any of the regions required for male fertility. In contrast to D. melanogaster, where Y-linked copies were found in nine different wild-type strains, no Y-linked copies were found in sibling species. Several apparently Y-derived cDNA clones and one Y-linked genomic clone have been sequenced. The Y-derived genomic DNA shares the same intron/exon structure as the autosomal copy as well as related flanking sequences suggesting that it transposed to the Y from the autosomal locus. However, this particular Y-linked copy cannot encode a functional polypeptide due to a stop codon at amino acid position 72. Divergence among five different cDNA clones ranges from 1.5 to 6% and includes a large number of third position substitutions. We have not yet obtained a full-length cDNA from a Y-linked gene and therefore cannot conclude that the D. melanogaster Y chromosome contains functional protein-coding genes. The autosomal gene encodes a predicted polypeptide with 45% similarity to histones of the H5 class and more limited similarity to cysteine-rich protamines. This protein may be a distant relative of the histone H1 family perhaps involved in sperm chromatin condensation.

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39 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....
[...]
...It has been proposed that Mst77F is essential for male fertility (Rathke et al. 2010), but this conclusion is based on the analysis of the antimorphic point mutation Mst77F1 (see Krsticevic et al. 2010)....
[...]
...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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...In addition, we confirmed that a transgene expressing Mst77FEGFP was normally incorporated into the chromatin of mutant gametes but failed to rescue the phenotype (Figure 3B)....
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...Interestingly, the D. melanogaster genome contains several recent copies of Mst77F on the Y chromosome, and eight of these Mst77Y genes are most likely functional (Russell and Kaiser 1993; Krsticevic et al. 2010)....
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