InteractiveFly: GeneBrief

deadhead: Biological Overview | References

Gene name - deadhead

Synonyms - Trx-1

Cytological map position - 1-11

Function - enzyme

Keywords - ovary-specific thioredoxin - linked to the distinctive redox-state balance set at the oocyte-to-embryo transition - redox proteome - required to unlock sperm chromatin at fertilization - disulfide bond reduction is required for protamine eviction from sperm chromatin

Symbol - dhd

FlyBase ID: FBgn0011761

Genetic map position - chrX:5,312,171-5,313,122

NCBI classification - TRX_family

Cellular location - cytoplasmic

NCBI link: EntrezGene, Nucleotide, Protein

dhd orthologs: Biolitmine
Recent literature
Torres-Campana, D., Kimura, S., Orsi, G. A., Horard, B., Benoit, G. and Loppin, B. (2020). The Lid/KDM5 histone demethylase complex activates a critical effector of the oocyte-to-zygote transition. PLoS Genet 16(3): e1008543. PubMed ID: 32134927
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. This study performed an shRNA-based genetic screen in Drosophila to identify genes required to form a diploid zygote. 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. It is proposed 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.
Torres-Campana, D., Horard, B., Denaud, S., Benoit, G., Loppin, B. and Orsi, G. A. (2022). Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain. PLoS Genet 18(1): e1009615. PubMed ID: 34982772
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. This study 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, it was 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, it was shown that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, this 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.

The metabolic and redox state changes during the transition from an arrested oocyte to a totipotent embryo remain uncharacterized. This study applied state-of-the-art, integrated methodologies to dissect these changes in Drosophila. Early embryos were shown to have a more oxidized state than mature oocytes. Specific alterations were identified in reactive cysteines at a proteome-wide scale as a result of this metabolic and developmental transition. Consistent with a requirement for redox change, a role was demonstrated for the ovary-specific thioredoxin Deadhead (DHD). dhd-mutant oocytes are prematurely oxidized and exhibit meiotic defects. Epistatic analyses with redox regulators link dhd function to the distinctive redox-state balance set at the oocyte-to-embryo transition. Crucially, global thiol-redox profiling identified proteins whose cysteines became differentially modified in the absence of DHD. These potential DHD substrates were validated by recovering DHD-interaction partners using multiple approaches. One such target, NO66, is a conserved protein that genetically interacts with DHD, revealing parallel functions. As redox changes also have been observed in mammalian oocytes, a link between developmental control of this cell-cycle transition and regulation by metabolic cues is hypothesized. This link likely operates both by general redox state and by changes in the redox state of specific proteins. The redox proteome defined here is a valuable resource for future investigation of the mechanisms of redox-modulated control at the oocyte-to-embryo transition (Petrova, 2018).

Although life is driven by reduction-oxidation (redox) reactions, remarkably little is known about how metabolic state interfaces with normal development. Reactive oxygen species (ROS) were first described as a byproduct of metabolism and a hallmark of disease and aging. Exciting new research, however, implicated ROS more directly in cell signaling and regulation. In a manner analogous to posttranslational modifications, ROS can alter the oxidation status of cysteine residues and thus affect protein stability, activity, and localization or protein-protein interactions. In this respect, how ROS link to the spatiotemporal regulation of development via downstream targets has not been explored thoroughly (Petrova, 2018).

The redox state in the cell exists in a dynamic balance between ROS production and removal. The source of ROS is primarily oxidative phosphorylation in mitochondria. The antioxidants catalase and superoxide dismutase (SOD) neutralize ROS in a direct enzymatic reaction. Further major ROS scavenger systems are glutathione (GSH), which exists as GSH in its reduced state and as glutathione disulfide (GSSG) in its oxidized state, and thioredoxins. GSH and thioredoxin utilize reducing power provided by oxidative metabolism, tied to redox couples such as NADPH/NADP+, to counteract oxidation via ROS. Under the redox-optimized ROS balance hypothesis, energy metabolism, antioxidants, and redox couples are in equilibrium to allow physiological ROS signaling under varying conditions. Ultimately, the redox balance sets the oxidation level of downstream targets via an interplay of scavenger systems with downstream substrates such as protein and lipids (Petrova, 2018).

A key developmental event is the oocyte-to-embryo transition, when at fertilization the highly specialized oocyte becomes a totipotent embryo. The oocyte-to-embryo transition, occurring in the absence of transcription, relies on posttranscriptional and posttranslational control. In this respect, could redox modulation of protein function bring a highly dynamic, additional level of regulation? Indeed, Dumollard (2008) showed that metabolic activity and thus the redox potential varies during mouse oogenesis and fertilization and is critical for early development. In addition, several aspects of oogenesis and early embryogenesis were blocked if proper redox balance was altered. Although these studies suggest a direct role for ROS, no comprehensive analysis has been carried out, and the downstream targets have not been investigated rigorously (Petrova, 2018).

Intriguingly, Drosophila expresses an oocyte-specific thioredoxin, Deadhead (DHD). DHD is required for early embryogenesis and timely protamine-to-histone exchange in the male pronucleus in fertilized eggs. DHD redox activity is essential for its function. A further exciting observation was that a ubiquitous thioredoxin, Trx-2, did not recognize protamines as substrates. This indicates that DHD has at least one specific target in early development. Because the protamine exchange defect did not fully account for the developmental block in the mutant, more roles and thus additional specific substrates are postulated for the functions DHD likely controls through redox regulation in the oocyte-to-embryo transition in Drosophila (Petrova, 2018).

This study describes an integrated approach to understand the role of redox in oogenesis and early embryogenesis in Drosophila. Proper redox balance is shown to be essential for meiosis completion, fertilization, and early embryogenesis. Use was made of recent technological breakthroughs enabling quantitative examination of the redox Cys-proteome. The data corroborate the emerging view that redox systems selectively maintain a nonequilibrium redox dynamic in distinct sets of the redox Cys-proteome. It is proposed that the developmentally regulated thioredoxin DHD controls a specific set of substrates by interpreting global changes at the level of energy metabolism. These findings provide a paradigm for understanding redox-sensitive targets and pathways in other organisms or systems where cell-state transitions are accompanied by global metabolic remodeling (Petrova, 2018).

Although redox state changes have been described to occur in oogenesis and embryogenesis in diverse systems, no comprehensive analysis of the regulators and targets of redox has been carried out. This work demonstrates that global redox changes occur at the oocyte-to-embryo transition in Drosophila. Redox pair measurements by LC-MS or in vivo H2O2 sensor imaging showed that embryos are more oxidized. The developmentally regulated thioredoxin DHD contributes to these changes so that the GSH/GSSG and NADH/NAD+ ratios and H2O2 levels in oocytes partially depend on its proper function. DHD works in conjunction with other redox proteins to ensure the fidelity of meiosis completion, fertilization, and early embryogenesis. DHD likely exerts its function via specific substrates, and thie study found that DHD interacts with other proteins in addition to the previously described protamines. Importantly, global changes in redox state were linked to corresponding changes in the redox-thiol proteome, and a set of specific DHD-dependent redox targets were documented. One such substrate, NO66, contributes to DHD function during early embryogenesis. This study leads to the hypothesis that metabolic changes occurring in the context of a developmental transition can be utilized to modify the function of specific downstream proteins and protein networks via dedicated redox systems (Petrova, 2018).

These work extends previous studies describing metabolic changes in Drosophila during oogenesis and embryogenesis. The metabolome changes as the Drosophila fertilized oocyte is activated to proceed through embryonic development. Within a very short time frame, 0-1 h after egg-laying, there are metabolic changes that affect, among others, the TCA cycle, amino acid metabolism, the pentose phosphate pathway, and purine metabolism. Although the mechanism by which metabolic changes and redox interact in early embryogenesis in Drosophila is yet to be determined, it is possible that this could occur via modulation of mitochondrial functions. Recently, mitochondria were shown to exist in a quiescent state in Drosophila oocytes and to reactivate in embryos. It will be interesting to investigate in the future how mitochondrial function, calcium signaling, and ROS generation and signaling are coupled at the oocyte-to-embryo transition (Petrova, 2018).

The oocyte-specific thioredoxin DHD is a key developmentally regulated node in the redox control of the oocyte-to-embryo transition in Drosophila. DHD protein levels are regulated, increasing markedly during oocyte maturation and sharply decreasing at egg activation. Interestingly, sperm-specific thioredoxins have been described in mammals as well as in Drosophila. Developmental control of thioredoxins could thus be a broader strategy to modulate cell-state transitions in other systems (Petrova, 2018).

A key question that this study poses is whether DHD exerts its essential roles at the oocyte-to-embryo transition via modulation of global redox state and/or via specific downstream targets. This study found that DHD is partially required for redox state in late oocytes. The effect on the GSH/GSSG ratio could be via thioredoxin's direct contribution in Drosophila to the GSH cycle, as no GSH reductase has been found. Interestingly, genetic analysis of interaction between dhd and trx-2 revealed an overlap of function, such that the proteins could cooperate in setting the proper GSH/GSSG ratio in late oocytes and early embryos. The genetic interactions between dhd and other genes involved in redox control argue for a global role for general redox in the oocyte-to-embryo transition. This conclusion is supported further by the observation that modulation of the redox state balance by the addition of DTT affected progression through meiosis in the in vitro system (Petrova, 2018).

In addition to redox control at a global level, this study showed both by physical interactions and reactive-thiol mass spectroscopy profiling that specific proteins are DHD targets. Along with TrxR1, a well-known DHD partner, NO66 was one of the strongest interactors. NO66 was identified by thiol-redox proteomics analysis as having a cysteine residue (C239) whose reactivity diminished, indicating thiol-oxidation, in dhdJ5/dhdP8-mutant embryos. NO66 is a JmjC domain-containing histone demethylase, shown recently to act as a suppressor of variegation and to localize to the nucleolus in flies. A link to redox control is that JmjC domain-containing proteins have been described as requiring ascorbate, a reducing agent, for optimal activity in vitro. The colocalization between DHD and NO66 in the nucleolus in S2 cells is consistent with a functional relationship. Importantly, the genetic interaction between no66 and dhd further supported this notion. DHD interacts with and/or modulates ribosomal and RNA-binding proteins and thus could control aspects of nucleolar or ribosomal function. Interestingly, the human NO66 has been described as having ribosome oxygenase activity (44). In the future, it will be important to mutate redox-sensitive cysteine residues on potential DHD targets and examine functions in vivo (Petrova, 2018).

Intriguingly, the mitochondrial NADH dehydrogenase ND-75 is among the top changed reactive cysteine-containing proteins in dhd mutants. Although the same residue (C193) is not present in the wild-type reactive Cys-proteome, the conserved cysteine Cys712 changes reactivity between oocytes and embryos. As ND-75, along with other complex I subunits, leaks into the cytoplasm in late-stage oocytes, an exciting possibility is that DHD-dependent global redox changes occur via modulation of mitochondrial metabolism. Furthermore, in an interaction partner analysis, the presence of SOD1, a thioredoxin partner previously described in plants, was noted. These observations suggest that the effect of DHD on global H2O2 as well as on NADH/NAD+ levels could be via modulation of the function of downstream targets. Thus, global and specific redox roles are likely interwoven. Overall, a model is proposed whereby DHD affects specific targets in a developmental context, thus 'translating' the metabolic and redox changes to control important regulators of the oocyte-to-embryo transition (Petrova, 2018).

Thiol-redox proteomic studies have been widely successful in understanding thiol-redox dynamics in various systems but have not been broadly applied in a developmental context. In this regard, model organisms offer the advantage of applying integrated approaches. For example, one study monitored thiol-redox changes as a function of life span in Caenorhabditis elegans. Another showed the dramatic effect of fasting on the Drosophila reactive-thiol proteome. This study demonstrates the use of thiol-redox proteomics to investigate the oocyte-to-embryo transition in Drosophila. In addition to identifying specific targets of DHD, 500 cysteines were found to become differentially reactive at egg activation. This dataset will be a valuable resource for the community. The results provide evidence that a defined set of regulators along with general redox state control the oocyte-to-embryo transition. It is concluded that remodeling of the reactive thiol proteome should be considered a fundamental part of this critical developmental window (Petrova, 2018).

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

In most animals, the extreme compaction of sperm DNA is achieved after the massive replacement of histones with sperm nuclear basic proteins (SNBPs), such as protamines. In some species, the ultracompact sperm chromatin is stabilized by a network of disulfide bonds connecting cysteine residues present in SNBPs. Studies in mammals have established that the reduction of these disulfide crosslinks at fertilization is required for sperm nuclear decondensation and the formation of the male pronucleus. This study shows 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. DHD localizes to the sperm nucleus to reduce its disulfide targets and is then rapidly degraded after fertilization (Tirmarche, 2016).

In sexually reproducing animals, the differentiation of haploid spermatids into mature spermatozoa involves major reorganization of the nuclear architecture. Starting as round nuclei after the second male meiotic division, spermatid nuclei slowly transform to eventually acquire the final, species-specific shape of mature sperm nuclei. Extreme compaction of nuclear DNA generally accompanies the streamlining of spermatids, resulting in the shutdown of basic nuclear activities, including transcription and DNA repair. In most species, sperm nuclear compaction requires the massive replacement of somatic-type histones with sperm nuclear basic proteins (SNBPs). SNBPs encompass a heterogeneous group of chromosomal proteins that are specifically expressed in male germ cells and deposited during spermiogenesis. Protamines, the best characterized SNBPs, are small (50-60 aa), arginine rich proteins present in vertebrates and some invertebrates. A model based on mammalian protamines has proposed that these positively charged proteins bind the major groove of the double helix and form toroid-like structures containing about 50 kilobases of sperm DNA. Protamines in eutherian mammals and a few other animal groups are also enriched in cysteine residues, which are otherwise rare in chromosomal proteins, including histones and most SNBPs. During sperm maturation in eutherian mammals, oxidation of the protamine cysteine thiols (-SH) allows the formation of a tridimensional network of disulfide bridges (-S-S-). Intermolecular disulfide crosslinks notably participate in the stabilization of sperm chromatin by connecting adjacent chromatin fibres. It is actually well established that for most species of mammals, a thiol reducing agent, such as dithiothreitol (DTT), is required to elicit sperm nuclear decondensation in vitro. In addition, it has been alternatively proposed that in human sperm, protamines thiols are non-covalently bridged by Zinc (Zn2+), thereby preventing or limiting the formation of excess disulfide bonds that could perturb sperm nuclear decondensation at fertilization (Tirmarche, 2016).

This work functionally characterized the Drosophila maternal thioredoxin Deadhead (DHD) and we demonstrate that DHD is required to unlock sperm chromatin at fertilization. Thioredoxins are small redox proteins found in all organisms. They typically reduce disulfide bonds on target proteins using a pair of cysteine thiols present in their conserved active CGPC site. Thioredoxins play important metabolic, protective or signalling functions but the molecular bases of their target specificity or functional specialization remain poorly understood. Three classical thioredoxins are found in Drosophila melanogaster. Trx-2 is a non-essential ubiquitous protein that participates in the protection against oxidative stress, whereas TrxT and DHD are sex-specific thioredoxins encoded by a pair of adjacent genes. While the role of the testis-specific TrxT is unknown, DHD is specifically expressed in the female germline and is essential for embryo development. In this work, we demonstrate that DHD is essential for sperm nuclear decondensation at fertilization (Tirmarche, 2016).

The stabilization of sperm chromatin with disulfide bonds is a remarkable innovation that emerged independently in distantly-related animal groups with the selective acquisition of cysteine residues in SNBPs. This work first established that Drosophila sperm chromatin, which is packaged with cysteine-rich SNBPs, is stabilized by disulfide crosslinks. The reducing agent DTT allows efficient decompaction of Drosophila sperm nuclei in vitro, in a way similar to its well-established activity on sperm nuclei from eutherian mammals, for instance. Similarly, it has been recently shown that pre-treatment of Drosophila sperm nuclei with DTT improves antibody accessibility to nuclear epitopes in immunofluorescence experiments. Although little is known about the structural organization of Drosophila sperm chromatin, a recent study showed that the Drosophila SNBP Mst77F is a DNA binding protein which induces DNA condensation in vitro through a multimerization process involving its coiled-coil domain. Oxidation of cysteine thiols present in Mst77F could participate in the stabilization of this unique chromatin architecture through the establishment of disulfide crosslinks. Disulfide bonds could also allow the formation of inter-chromatin fibre associations, as proposed for mammalian sperm chromatin (Tirmarche, 2016).

The implication of DHD in the reduction of sperm chromatin disulfide crosslinks in Drosophila eggs is first supported by a detailed characterization of the dhd phenotype. Among the rare Drosophila maternal effect mutants affecting sperm chromatin remodelling, dhd is unique and shows the earliest phenotype reported so far. Notably, in dhd mutant eggs, the sperm nucleus is initially intact, showing no sign of decondensation. Second, any role of DHD in the reduction of sperm nuclear disulfide bonds implies the critical requirement of its catalytic redox motif. Previous work has demonstrated that transgenes expressing DHD in which the two active cysteines are replaced by serines failed to rescue the fertility of mutant females. This work has shown that replacing the second cysteine residue of the redox motif not only abolished DHD function but also trapped the protein on sperm chromatin in a remarkably specific manner. This experiment strongly suggests that DHD directly targets disulfide bonds on sperm chromatin. Interestingly, the DHDC34S mutant protein could not rescue the dhd nuclear phenotype despite its expected ability to cleave its disulfide targets. It is speculated that the irreversible covalent trapping of DHDC34S on SNBPs at the surface of sperm chromatin could impede their eviction and prevent further access of the thioredoxin to the compact sperm nucleus. It is proposed that, following sperm penetration through the egg micropyle and sperm plasma membrane breakdown, the sperm nucleus is exposed to the egg cytoplasm that contains massive amounts of DHD thioredoxin. The reduction of disulfide crosslinks by DHD induces sperm nuclear decompaction and allows the subsequent extraction of SNBPs from sperm chromatin, a process that likely involves the ISWI chromatin remodeler. Finally, the removal of SNBP leaves DNA accessible to the HIRA complex for genome-wide nucleosome assembly with maternally supplied histones (Tirmarche, 2016).

In mammals, the tripeptide antioxidant glutathione is abundant in oocytes and plays an important role in sperm nuclear decondensation. Inhibition of glutathione synthesis by buthionine sulfoximine during oocyte maturation indeed efficiently blocks sperm nuclear decondensation and this effect can be reversed by DTT. Whether glutathione directly reduces SNBP disulfides in vivo or instead activates a dedicated disulfide reductase is still unresolved. In most species, glutathione disulfide (GSSG) is reduced into glutathione (GSH) by the highly conserved glutathione reductase enzyme. Drosophila is unusual in lacking glutathione reductase and instead relies on the thioredoxin system for GSSG reduction. Interestingly, biochemical characterization of Drosophila thioredoxins have established that DHD and Trx-2 are equally efficient for GSSG reduction in vitro, opening the possibility that DHD could indirectly influence sperm nuclear decondensation by controlling the level of GSH in eggs. However, the demonstration that recombinant DHD protein is sufficient for sperm nuclear decondensation and SNBP removal in an in vitro assay argues against this hypothesis. Still, this study has clearly documented that sperm chromatin remodelling is not completely blocked in dhd eggs but instead occurs very slowly. This slow replacement of SNBPs with histones suggests that mutant eggs still have residual reducing power for the cleavage of SNBP disulfide bonds, an effect that could likely involve glutathione itself (Tirmarche, 2016).

This study also revealed that the migration of the female pronucleus to the centre of the egg fails in a majority of dhd eggs, thus preventing the initiation of embryo development. The release of paternal centrioles from the base of the sperm nucleus and the subsequent elaboration of the sperm aster were indeed affected in mutant eggs. As a matter of fact, depletion of glutathione in bovine oocytes not only blocks sperm nuclear decondensation but also prevents sperm aster growth and pronuclear migration. It was observed that the disassembly of the sperm tail and connecting piece, as well as the release of the proximal centriole were frequently affected in treated oocytes, suggesting that the role of glutathione at fertilization is not restricted to sperm chromatin decompaction. Further investigation will be required to determine whether DHD similarly targets additional sperm structures (Tirmarche, 2016).

In conclusion, it is proposed that the rapid unlocking of sperm chromatin at fertilization in Drosophila is critically dependent on the redox activity of the egg specific DHD thioredoxin. dhd is apparently the most recent thioredoxin gene in Drosophila and likely originated after the duplication of the ancestral Trx-2 gene. It is thus tempting to propose that the emergence of this highly specialized protein could reflect the adaptation of the oocyte to the rapid evolution of sperm chromatin architecture. Future work will aim at understanding how the structural features of DHD underlie its crucial specialization at the onset of embryo development (Tirmarche, 2016).

Thioredoxin-dependent disulfide bond reduction is required for protamine eviction from sperm chromatin

Cysteine oxidation in protamines leads to their oligomerization and contributes to sperm chromatin compaction. This study identifies the Drosophila thioredoxin Deadhead (DHD) as the factor responsible for the reduction of intermolecular disulfide bonds in protamines and their eviction from sperm during fertilization. Protamine chaperone TAP/p32 dissociates DNA-protamine complexes in vitro only when protamine oligomers are first converted to monomers by DHD. dhd-null embryos cannot decondense sperm chromatin and terminate development after the first pronuclear division. Therefore, the thioredoxin DHD plays a critical role in early development to facilitate the switch from protamine-based sperm chromatin structures to the somatic nucleosomal chromatin (Emelyanov, 2016).

The amorphic mutant of Drosophila deadhead (dhd) has been described. dhd is not essential for adult viability but is recessive maternal effect lethal: The majority of eggs laid by homozygous females is fertilized but fails to initiate development. To test whether DHD affects sperm decondensation and protamine eviction, homozygous dhd mothers were crossed with fathers that carry transgenes expressing eGFP-tagged ProtB and don juan (dj) that encode the major components of sperm heads and tails, respectively. Heterozygous dhd/FM7 mothers were used in control crosses. dhd embryos were completely incapable of processing sperm chromatin. Microscopic analyses revealed that the majority of 0- to 4-h embryos (>60% of the total scored) was fertilized and contained GFP-labeled sperm. Importantly, they failed to remove Prot B from sperm heads, which remained fully compacted. In most of the embryos (~55% of the total), the female pronuclei underwent one haploid mitosis but terminated further divisions. The sperm heads did not specifically migrate to the middle of the embryo but rather assumed random positions within the egg. In contrast, dhd/FM7 embryos did not exhibit any developmental defects. Also, no Prot B-eGFP-labeled sperm heads were observed after inspecting >2000 fertilized heterozygous embryos, consistent with an extremely fast (within minutes and, frequently, before egg deposition) sperm decondensation and protamine eviction during normal development (Emelyanov, 2016).

Occasionally, dhd/dhd embryos (~5% of the total) entered syncytial divisions but aborted their development prior to cellularization. Although persistent sperm cells were not detected in these syncytial embryos, other evidence indicated that they did not remodel sperm chromatin or form the male pronucleus. First, the appearance of anaphase chromosomes suggested a haploid DNA content. Furthermore, PCR analyses of maternal- and paternal-derived sequences in the genomic DNA of dhd embryos exposed a very strong overabundance of maternal DNA. The aborted development of gynogenetic haploid embryos in the dhd mutant is similar to that in mutants of ssm, yem, and Chd1, which encode the HIRA-YEM complex and CHD1, the factors required for nucleosome assembly in the male pronucleus. However, in contrast to dhd mutation, ssm, yem, and Chd1 mutations lead to the vast majority of embryos entering haploid syncytial divisions. Therefore, although DHD is clearly required for sperm chromatin remodeling in vivo, it may also be involved in other embryonic functions, such as regulation of DNA synthesis or S-phase initiation during preblastoderm mitosis, as proposed previously (Emelyanov, 2016).

In metazoan development, nuclear DNA undergoes dramatic differentiation-dependent, activity-dependent, and cell cycle-dependent transitions that alter the composition, distribution, and modification status of associated proteins. This study demonstrates that Drosophila sperm chromatin compaction involves oligomerization of protamines via intermolecular disulfide bridges. To convert the condensed, static, and metabolically inert paternal chromatin into a transcriptionally and otherwise enzymatically competent somatic cell chromatin, the embryo expresses a network of specialized proteins, which includes the thioredoxin DHD and protamine chaperones. Synergistically, they reverse the protamine oligomerization and remove them from DNA during fertilization. This network of physically interacting proteins plays an essential role in early embryonic development. Metazoans exhibit a strong similarity in amino acid content (cysteine enrichment) and secondary structure of protamines (intramolecular and intermolecular disulfide bonds) as well as primary structures of protamine chaperones and various thioredoxins. Thus, the function of the thioredoxin system in sperm chromatin remodeling is likely conserved in evolution (Emelyanov, 2016).

Organization and regulation of sex-specific thioredoxin encoding genes in the genus Drosophila

Thioredoxins are small thiol proteins that have a conserved active site sequence, WCGPC, and reduce disulfide bonds in various proteins using the two active site cysteines, a reaction that oxidizes thioredoxin and renders it inactive. Thioredoxin reductase returns thioredoxin to its reduced, active form in a reaction that converts NADPH to NADP(+). The biological functions of thioredoxins vary widely; they have roles in oxidative stress protection, act as electron donors for ribonucleotide reductase, and form structural components of enzymes. To date, three thioredoxin genes have been characterized in Drosophila melanogaster: the generally expressed Thioredoxin-2 (Trx-2) and the two sex-specific genes ThioredoxinT (TrxT) and deadhead (dhd). The male-specific TrxT and the female-specific dhd are located as a gene pair, transcribed in opposite directions, with only 470 bp between their transcription start points. This study shows that all three D. melanogaster thioredoxins are conserved in 11 other Drosophilid species, which are believed to have diverged up to 40 Ma ago and that Trx-2 is conserved all the way to Tribolium castaneum. This study has found that the intriguing gene organization and regulation of TrxT and dhd is remarkably well conserved, and potential conserved regulatory sequences were identified. In addition, this study shows that the 50-70 C terminal amino acids of TrxT constitute a hyper-variable domain, which could play a role in sexual conflict and male-female co-evolution (Svensson, 2007b).

Thioredoxin-2 affects lifespan and oxidative stress in Drosophila

Thioredoxins are proteins that have thiol-reducing activity and a characteristic conserved active site (WCGPC). They have several documented functions, e.g. roles in defences against oxidative stress and as electron donors for ribonucleotide-reductase. In Drosophila melanogaster there are three 'classical' thioredoxins with the conserved active site: deadhead, ThioredoxinT and Thioredoxin-2. This paper reports the creation of null-mutations in the Thioredoxin-2 (Trx-2) gene. Characterization of two Trx-2 mutants indicated that Trx-2 affects the lifespan of D. melanogaster, and is involved in the organism's oxidative stress protection system. The mutants have a shorter lifespan than wild-type flies, and thioredoxin double mutant flies showed lower tolerance to oxidative stress than wild-type flies, while flies carrying multiple copies of a Trx-2 rescue construct showed higher tolerance. These findings suggest that Trx-2 has modest or redundant functions in Drosophila physiology under unstressed conditions, but could be important during times of environmental stress (Svensson, 2007a).

Expression of meiotic genes in the germline progenitors of Drosophila embryos

Meiosis is one of the fundamental characteristics of germ cells. In Drosophila, genetic screens have identified many genes required for meiotic division. However, it remains elusive as to when and how these meiotic genes are activated during germline development. To obtain insights into their regulatory mechanisms, this study examined the expression of 38 meiotic genes in the germline progenitors, pole cells, during embryogenesis. The transcripts of 12 meiotic genes were enriched in pole cells within the embryonic gonads. Among them, bag of marbles (bam), benign gonial cell neoplasia (bgcn), deadhead (dhd), matotopetli (topi) and twine (twe) were activated only in pole cells within the gonads, whereas the transcripts from grapes (grp), Kinesin-like protein at 3A (Klp3A), pavarotti (pav), lesswright (lwr), mei-P26, Topoisomerase 2 (Top2) and out at first (oaf) were distributed ubiquitously in early embryos and then became restricted to pole cells and to a subset of somatic tissues at later embryonic stages. The remaining meiotic genes were either expressed ubiquitously in the embryos (15 genes) or were undetectable in pole cells within the gonads (11 genes). These observations suggest that pole cells have already acquired the potential to express several meiotic genes. These data will thus provide a useful basis for analyzing how the germline acquires a potential to execute meiosis (Mukai, 2006).

Regulatory role of dADAR in ROS metabolism in Drosophila CNS

Pre-mRNA adenosine deaminase (ADAR) is involved in many physiological processes by either directly converting adenosine to inosine in certain pre-mRNAs or indirectly regulating expression of certain genes. Mutations of Drosophila ADAR (dADAR) results in neuronal dysfunction and hypersensitivity to oxygen deprivation. Recently, it was found that the mutant flies were very resistant to paraquat, a compound that generates free radicals. In order to further characterize the neuronal role of dADAR and understand the basis for the resistance to the oxidative stress, this study investigated the effect of dADAR on the expression of genes encoding scavengers of cellular reactive oxygen species (ROS) in both dADAR mutant and overexpression flies. The data show that the expression of the genes encoding known ROS scavengers [superoxide dismutase (SOD) and catalase] is not regulated by dADAR. However, the transcripts of genes encoding two potential ROS scavengers (dhd and Cyp4g1) were robustly increased in dADAR mutant flies, and conversely both were significantly decreased in dADAR overexpressing flies. Using dhd [encoding a Drosophila homolog of the mammalian protein thioredoxin (Trx)] transgenic flies, it was confirmed that the resistance of dADAR mutant flies to paraquat resulted, at least partially, from the up-regulation of dhd gene in dADAR mutant flies. These data not only confirm the importance of ADAR in maintenance of neuronal function but also reveal its regulatory role in the expression of genes encoding ROS scavengers (Chen, 2004).

The ThioredoxinT and deadhead gene pair encode testis- and ovary-specific thioredoxins in Drosophila melanogaster

So far, two thioredoxin proteins, DHD and Trx-2, have been biochemically characterized in Drosophila melanogaster. With the cloning and characterization of TrxT this study describes an additional thioredoxin with testis-specific expression. TrxT and dhd are arranged as a gene pair, transcribed in opposite directions and sharing a 471 bp regulatory region. This regulatory region is sufficient for correct expression of the two genes. This gene pair makes a good model for unraveling how closely spaced promoters are differentially regulated by a short common control region. Both TrxT and DHD proteins are localized within the nuclei in testes and ovaries, respectively. Use of a transgenic construct expressing TrxT fused to Enhanced Yellow Fluorescent Protein reveals a clear association of TrxT with the Y chromosome lampbrush loops ks-1 and kl-5 in primary spermatocytes. The association is lost in the absence of the Y chromosome. These results suggest that nuclear thioredoxins may have regulatory functions in the germline (Svensson, 2003).

Thioredoxin-2 but not thioredoxin-1 is a substrate of thioredoxin peroxidase-1 from Drosophila melanogaster: isolation and characterization of a second thioredoxin in D. melanogaster and evidence for distinct biological functions of Trx-1 and Trx-2

As Drosophila melanogaster does not contain glutathione reductase, the thioredoxin system has a key function for glutathione disulfide reduction in insects. In view of these unique conditions, the protein systems participating in peroxide metabolism and in redox signaling are of special interest. The genes for a second thioredoxin (DmTrx-2) and a thioredoxin peroxidase (DmTPx-1) were cloned and expressed, and the proteins were characterized. In its disulfide form, the 13-kDa protein thioredoxin-2 is a substrate of thioredoxin reductase-1 and in its dithiol form, an electron donor for TPx-1. DmTrx-2 is capable of reducing glutathione disulfide at pH 7.4 and 25 degrees C. Western blot analysis indicated that this thioredoxin represents up to 1% of the extractable protein of D. melanogaster Schneider cells or whole fruit flies. Recombinant thioredoxin peroxidase-1 (subunit molecular mass = 23 kDa) was found to be a decameric protein that can efficiently use Trx-2 but not Trx-1 as a reducing substrate. The new electron pathway found in D. melanogaster is also representative for insects that serve as vectors of disease. As a first step this study has cloned and functionally expressed the gene that is the orthologue of DmTrx-2 in the malaria mosquito Anopheles gambiae (Bauer, 2002).

The function of the Drosophila thioredoxin homologue encoded by the deadhead gene is redox-dependent and blocks the initiation of development but not DNA synthesis

Thioredoxin is a highly conserved disulfide reducing protein whose structure and biochemical properties have been extensively studied. Nonetheless, its function in vivo is not well defined. In Drosophila, the maternal-effect gene deadhead encodes a thioredoxin-like protein that is required for initiation of embryonic development. This study reports that deadhead function is dependent on its enzymatic activity: transgenes carrying mutations in thioredoxin's conserved active site failed to rescue the deadhead mutant phenotype. A number of studies have documented that thioredoxin plays a role in DNA synthesis. If thioredoxin is required for DNA synthesis in the fly, then deadhead mutations will suppress mutations that inappropriately synthesize DNA. Contrary to expectation, this study found that deadhead does not function as a suppressor in this assay. The observed epistatic relationship between these mutations clearly indicates that deadhead is not essential for DNA metabolism. The possibility of a regulatory role in controlling the initiation of S-phase is discussed (Pellicena-Palle, 1997).

The Drosophila maternal effect locus deadhead encodes a thioredoxin homolog required for female meiosis and early embryonic development

This study describes the identification, function and molecular characterization of deadhead, a Drosophila thioredoxin homolog. Although in vitro studies have shown that thioredoxin can post-translationally regulate the activity of many different proteins, this study found that this homolog is not essential for viability. The phenotypic analysis of two different mutations which eliminate function suggests that dhd is essential for female meiosis. The majority of eggs laid by females homozygous for null mutations are fertilized but fail to complete meiosis. A small number of escaper embryos initiate development and display a range of phenotypes suggesting functions in both preblastoderm mitosis and head development. This analysis of deadhead RNA expression pattern is consistent with its maternal effect function: the RNA is predominately expressed in the nurse cells of the ovary, is maternally deposited into the egg, but does not appear to be zygotically expressed during embryogenesis. Thus both genetic and molecular data are consistent with a function during meiosis and preblastoderm mitosis. Whether the head defect indicates an additional function or is an indirect consequence of earlier defects remains to be determined (Salz, 1994).


Search PubMed for articles about Drosophila Deadhead

Bauer, H., Kanzok, S. M. and Schirmer, R. H. (2002). Thioredoxin-2 but not thioredoxin-1 is a substrate of thioredoxin peroxidase-1 from Drosophila melanogaster: isolation and characterization of a second thioredoxin in D. Melanogaster and evidence for distinct biological functions of Trx-1 and Trx-2. J Biol Chem 277(20): 17457-17463. PubMed ID: 11877442

Chen, L., Rio, D. C., Haddad, G. G. and Ma, E. (2004). Regulatory role of dADAR in ROS metabolism in Drosophila CNS. Brain Res Mol Brain Res 131(1-2): 93-100. PubMed ID: 15530657

Dumollard, R., Campbell, K., Halet, G., Carroll, J. and Swann, K. (2008). Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes. Dev Biol 316(2): 431-440. PubMed ID: 18342302

Emelyanov, A. V. and Fyodorov, D. V. (2016).Thioredoxin-dependent disulfide bond reduction is required for protamine eviction from sperm chromatin. Genes Dev [Epub ahead of print]. PubMed ID: 28031247

Mukai, M., Kitadate, Y., Arita, K., Shigenobu, S. and Kobayashi, S. (2006). Expression of meiotic genes in the germline progenitors of Drosophila embryos. Gene Expr Patterns 6(3): 256-266. PubMed ID: 16412701

Pellicena-Palle, A., Stitzinger, S. M. and Salz, H. K. (1997). The function of the Drosophila thioredoxin homologue encoded by the deadhead gene is redox-dependent and blocks the initiation of development but not DNA synthesis. Mech Dev 62(1): 61-65. PubMed ID: 9106167

Petrova, B., Liu, K., Tian, C., Kitaoka, M., Freinkman, E., Yang, J. and Orr-Weaver, T. L. (2018). Dynamic redox balance directs the oocyte-to-embryo transition via developmentally controlled reactive cysteine changes. Proc Natl Acad Sci U S A 115(34): E7978-E7986. PubMed ID: 30082411

Salz, H. K., Flickinger, T. W., Mittendorf, E., Pellicena-Palle, A., Petschek, J. P. and Albrecht, E. B. (1994). The Drosophila maternal effect locus deadhead encodes a thioredoxin homolog required for female meiosis and early embryonic development. Genetics 136(3): 1075-1086. PubMed ID: 7516301

Svensson, M. J., Chen, J. D., Pirrotta, V. and Larsson, J. (2003). The ThioredoxinT and deadhead gene pair encode testis- and ovary-specific thioredoxins in Drosophila melanogaster. Chromosoma 112(3): 133-143. PubMed ID: 14579129

Svensson, M. J. and Larsson, J. (2007a). Thioredoxin-2 affects lifespan and oxidative stress in Drosophila. Hereditas 144(1): 25-32. PubMed ID: 17567437

Svensson, M. J., Stenberg, P. and Larsson, J. (2007b). Organization and regulation of sex-specific thioredoxin encoding genes in the genus Drosophila. Dev Genes Evol 217(9): 639-650. PubMed ID: 17701050

Tirmarche, S., Kimura, S., Dubruille, R., Horard, B. and Loppin, B. (2016). Unlocking sperm chromatin at fertilization requires a dedicated egg thioredoxin in Drosophila. Nat Commun 7: 13539. PubMed ID: 27876811

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date revised: 22 February 2022

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