Male-specific Transcription Research Articles

Differentially expressed genes in Jojoba (Simmondsia chinensis) when subjected to water-stress

Jojoba (Simmondsia chinensis) is a desert shrub with an ability to survive in extremely arid environments. The exceptional drought tolerance of jojoba includes traits that could be useful in developing drought tolerant crop plants. This study characterized changes in gene expression in jojoba under water-stress during a controlled-environment experiment. A large number of transcripts (10,936) were identified as differentially expressed under contrasting water stress conditions. These included transcripts corresponding to antioxidant activity-related genes such as superoxide dismutase, defense response genes such as Pathogenesis-Related 4 (PR4), water molecule bio-channels such as aquaporins, cell membrane protectants such as Late Embryogenesis Abundant (LEA), and growth regulators such as 1-aminocyclopropane-1-carboxylic acid (ACC). A total of 880 novel transcripts were identified as representing possible novel genes associated with jojoba subjected to water stress. There were also many transcripts linked to transcriptional regulation that were expressed in response to water-stress in jojoba. Many male-specific transcripts corresponding to stress-related genes and transcription regulators were differentially expressed under water-stress with 1928 differentially expressed transcripts that aligned to the two Y chromosome-specific regions Y1 and Y2. The water-stress related genes detected may help explain the drought tolerance of jojoba and provide a valuable source of genes for exploitation in providing tolerance to water stress in other species.

A male-specific doublesex isoform reveals an evolutionary pathway of sexual development via distinct alternative splicing mechanisms

The doublesex/mab-3 related transcription factor (Dmrt) genes regulate sexual development in metazoans. Studies of the doublesex (dsx) gene in insects, in particular Drosophila melanogaster, reveal that alternative splicing of dsx generates sex-specific Dsx isoforms underlying sexual differentiation. Such a splicing-based mechanism underlying sex-specific Dmrt function is thought to be evolved from a transcription-based mechanism used in non-insect species, but how such transition occurs during evolution is not known. Here we identified a male-specific dsx transcript (dsxM2) through intron retention (IR), in addition to previously identified dsxM and dsxF transcripts through alternative polyadenylation (APA) with mutually exclusive exons. We found that DsxM2 had similarly masculinizing function as DsxM. We also found that the IR-based mechanism generating sex-specific dsx transcripts was conserved from flies to cockroaches. Further analysis of these dsx transcripts suggested an evolutionary pathway from sexually monomorphic to sex-specific dsx via the sequential use of IR-based and APA-based alternative splicing.

Male-specific long non-coding RNA testis-specific transcript, Y-linked 15 promotes gastric cancer cell growth by regulating Wnt family member 1/β-catenin signaling by sponging microRNA let-7a-5p

ABSTRACT The present study is aimed to investigate the regulatory effects and related mechanism of long non-coding RNA testis-specific transcript, Y-linked 15 (TTTY15) in gastric carcinoma (GC) cell proliferation, migration, invasion, apoptosis and epithelial–mesenchymal transition (EMT). TTTY15 expression in GC tissue samples and cells was detected by quantitative real-time PCR (qRT-PCR), and the correlation between TTTY15 expression and GC clinicopathological indicators was analyzed. Cell counting kit-8 (CCK-8), BrdU, flow cytometry and Transwell assays were performed for detecting GC cell proliferation, migration, invasion and apoptosis. Western blot was performed for detecting the expressions of EMT-associated proteins (N-cadherin and E-cadherin), Wnt family member 1 (Wnt1) protein and β-catenin protein. Bioinformatics analysis was conducted to predict, and RNA immunoprecipitation (RIP) assay and dual-luciferase reporter gene assay were performed to verify the targeted relationships of microRNA let-7a-5p (let-7a-5p) with TTTY15 and Wnt1 mRNA 3'UTR. It was found that TTTY15 expression was significantly up-regulated in GC tissues and cells, and was associated with advanced TNM stage and poor tumor differentiation. TTTY15 overexpression promoted GC cell proliferation, migration and invasion, the expressions of N-cadherin, Wnt1 and β-catenin protein, and inhibited the apoptosis and E-cadherin expression, while knocking down TTTY15 had the opposite effects. TTTY15 directly targeted let-7a-5p and negatively regulated its expression. Wnt1 was the target gene of let-7a-5p, and TTTY15 could indirectly and positively regulate Wnt1 expression. In conclusion, TTTY15 promotes GC progression, by regulating the let-7a-5p/Wnt1 axis to activate the Wnt/β-catenin pathway.

CRISPR/Cas9 mediated sex-ratio distortion by sex specific gene editing in Aedes aegypti

Aedes aegypti is a principal vector for several viruses including dengue virus, chikungunya virus and zika virus. Economic burden of mosquito-borne diseases, relative failure of traditional control strategies and the resistance development against insecticides enforces towards genetic manipulation of Ae. aegypti. Hence, a key gene doublesex (Aedsx) which regulate sex differentiation and alternatively splices to form male and female specific transcripts (AedsxM and AedsxF). CRISPR/Cas9 technique was employed to sex specifically disrupt the female-specific isoforms, AedsxF1 and AedsxF2, both of which were shown to be expressed only in female mosquitoes. Targeting of dsxF at the developmental stage has resulted in various phenotypic anomalies of adult females. The rate of adult mutation phenotype was recorded between 29 and 37% along with anomalies of wing size, proboscis length and reduction in the sizes of pre-blood-meal and after blood-meal ovaries in dsxF1 and dsxF2 microinjected groups, respectively. These findings can be correlated with reduced fecundity rate of Go female, where AedsxF1 and AedsxF2 groups showed reduction rate in range of 23–31%. Furthermore, hatching inhibition rate of 28 to 36% was also observed in G1 generation when compared to the wildtype. Overall, these results demonstrated that AedsxF disruption has resulted in multiple female traits disruption including decreased fertility of the female that could directly or indirectly associated with reproduction and its disease transmitting abilities. All these findings suggesting that CRISPR works to alter the developmental pathways as predicted, and therefore this method potentially gives us the basis for the sex-ratio distortion system as genetic control approach for the management of this vector.

The glucose-sensing transcription factor MLX balances metabolism and stress to suppress apoptosis and maintain spermatogenesis.

Male germ cell (GC) production is a metabolically driven and apoptosis-prone process. Here, we show that the glucose-sensing transcription factor (TF) MAX-Like protein X (MLX) and its binding partner MondoA are both required for male fertility in the mouse, as well as survival of human tumor cells derived from the male germ line. Loss of Mlx results in altered metabolism as well as activation of multiple stress pathways and GC apoptosis in the testes. This is concomitant with dysregulation of the expression of male-specific GC transcripts and proteins. Our genomic and functional analyses identify loci directly bound by MLX involved in these processes, including metabolic targets, obligate components of male-specific GC development, and apoptotic effectors. These in vivo and in vitro studies implicate MLX and other members of the proximal MYC network, such as MNT, in regulation of metabolism and differentiation, as well as in suppression of intrinsic and extrinsic death signaling pathways in both spermatogenesis and male germ cell tumors (MGCTs).

Histone deacetylase inhibition leads to regulatory histone mark alterations and impairs meiosis in oocytes

BackgroundPanobinostat (PB), a histone deacetylase (HDAC) inhibitor drug, is clinically used in the treatment of cancers. We investigated the effects of PB on murine ovarian functions in embryos and adult animals.MethodsC57BL/6J mice were treated with 5 mg/kg PB on alternate days from embryonic day (E) 6.5 to E15.5. We analysed the effects of PB on the ovaries by using immunofluorescence, gene expression analysis and DNA methylation analysis techniques.ResultsAt E15.5, we observed increases in histone H3K9Ac, H4Ac and H3K4me3 marks, while the level of the silencing H3K9me3 mark decreased. Synaptonemal complex examination at E15.5, E17.5 and E18.5 showed a delay in meiotic progression characterized by the absence of synaptonemal complexes at E15.5 and the persistence of double-strand breaks (DSBs) at E17.5 and E18.5 in PB-exposed oocytes. We found that exposure to PB led to changes in the expression of 1169 transcripts at E15.5. Genes regulated by the male-specific factors SRY-Box Transcription Factor 9 (SOX9) and Doublesex and Mab-3-related Transcription factor 1 (DMRT1) were among the most upregulated genes in the ovaries of PB-exposed mice. In contrast, PB treatment led to decreases in the expression of genes regulated by the WNT4 pathway. Notably, we observed 119 deregulated genes encoding Zn-finger proteins. The observed alterations in epigenetic marks and gene expression correlated with decreases in the numbers of germ cells at E15.5. After birth, PB-exposed ovaries showed increased proliferation of primary and secondary follicles. We also observed decreases in the numbers of primordial, primary and secondary follicles in adult ovaries from mice that were exposed to PB in utero. Finally, epigenetic alterations such as decreased H3K4me3 and increased H4 acetylation levels were also detected in somatic cells surrounding fully grown oocytes.ConclusionOur data suggest that inhibition of histone deacetylase by PB during a critical developmental window affects reprogramming and germ cell specification via alteration of epigenetic marks.

Evolutionary transition of doublesex regulation from sex-specific splicing to male-specific transcription in termites

The sex determination gene doublesex (dsx) encodes a transcription factor with two domains, oligomerization domain 1 (OD1) and OD2, and is present throughout insects. Sex-specific Dsx splicing isoforms regulate the transcription of target genes and trigger sex differentiation in all Holometabola examined to date. However, in some hemimetabolous insects, dsx is not spliced sexually and its sequence is less conserved. Here, to elucidate evolutionary changes in dsx in domain organisation and regulation in termites, we searched genome and/or transcriptome databases for the dsx OD1 and OD2 in seven termite species and their sister group (Cryptocercus woodroaches). Molecular phylogenetic and synteny analyses identified OD1 sequences of termites and C. punctulatus that clustered with dsx of Holometabola and regarded them as dsx orthologues. The Cryptocercus dsx orthologue containing OD2 was spliced sexually, as previously shown in other insects. However, OD2 was not found in all termite dsx orthologues. These orthologues were encoded by a single exon in three termites for which genome information is available; they were not alternatively spliced but transcribed in a male-specific manner in two examined species. Evolution of dsx regulation from sex-specific splicing to male-specific transcription may have occurred at an early stage of social evolution in termites.

Alcohol-induced aggression in Drosophila.

Alcohol-induced aggression is a destructive and widespread phenomenon associated with violence and sexual assault. However, little is understood concerning its mechanistic origin. We have developed a Drosophila melanogaster model to genetically dissect and understand the phenomenon of sexually dimorphic alcohol-induced aggression. Males with blood alcohol levels of 0.04-mg/ml BAC were less aggressive than alcohol-naive males, but when the BAC had dropped to ~0.015 mg/ml, the alcohol-treated males showed an increase in aggression toward other males. This aggression-promoting treatment is referred to as the post-ethanol aggression (PEA) treatment. Females do not show increased aggression after the same treatment. PEA-treated males also spend less time courting and attempt to copulate earlier than alcohol-naive flies. PEA treatment induces expression of the FruM transcription factor (encoded by a male-specific transcript from the fruitless gene), whereas sedating doses of alcohol reduce FruM expression and reduce male aggression. Transgenic suppression of FruM induction also prevents alcohol-induced aggression. In male flies, alcohol-induced aggression is dependent on the male isoform of the fruitless transcription factor (FruM). Low-dose alcohol induces FruM expression and promotes aggression, whereas higher doses of alcohol suppress FruM and suppress aggression.

SNPC-1.3 is a sex-specific transcription factor that drives male piRNA expression in C. elegans.

Piwi-interacting RNAs (piRNAs) play essential roles in silencing repetitive elements to promote fertility in metazoans. Studies in worms, flies, and mammals reveal that piRNAs are expressed in a sex-specific manner. However, the mechanisms underlying this sex-specific regulation are unknown. Here we identify SNPC-1.3, a male germline-enriched variant of a conserved subunit of the small nuclear RNA-activating protein complex, as a male-specific piRNA transcription factor in Caenorhabditis elegans. SNPC-1.3 colocalizes with the core piRNA transcription factor, SNPC-4, in nuclear foci of the male germline. Binding of SNPC-1.3 at male piRNA loci drives spermatogenic piRNA transcription and requires SNPC-4. Loss of snpc-1.3 leads to depletion of male piRNAs and defects in male-dependent fertility. Furthermore, TRA-1, a master regulator of sex determination, binds to the snpc-1.3 promoter and represses its expression during oogenesis. Loss of TRA-1 targeting causes ectopic expression of snpc-1.3 and male piRNAs during oogenesis. Thus, sexually dimorphic regulation of snpc-1.3 expression coordinates male and female piRNA expression during germline development.

Targeting the autosomal Ceratitis capitata transformer gene using Cas9 or dCas9 to masculinize XX individuals without inducing mutations

BackgroundFemales of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs.ResultsWith the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults.ConclusionsOur data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination.

Distinct Roles and Synergistic Function of FruM Isoforms in Drosophila Olfactory Receptor Neurons

Sexual dimorphism in Drosophila courtship circuits requires the male-specific transcription factor fruM, which is alternatively spliced to encode the FruMA, FruMB, and FruMC isoforms. Most fruM-positive neurons express multiple variants; however, the functional significance of their co-expression remains undetermined. Do co-expressed isoforms each play unique roles to jointly regulate dimorphism? By focusing on fruM-positive olfactory receptor neurons (ORNs), here, we show that FruMB and FruMC are both required for males' age-dependent sensitization to aphrodisiac olfactory cues in a cell-autonomous manner. Interestingly, FruMB expression is upregulated with age in Or47b and Ir84a ORNs, and its overexpression mimics the effect of age in elevating olfactory responses. Mechanistically, FruMB and FruMC synergistically mediate response sensitization through cooperation of their respective downstream effectors, namely, PPK25 and PPK23, which are both required for forming a functional amplification channel in ORNs. Together, these results provide critical mechanistic insight into how co-expressed FruM isoforms jointly coordinate dimorphic neurophysiology.

Teiresias, a Fruitless target gene encoding an immunoglobulin-superfamily transmembrane protein, is required for neuronal feminization in Drosophila

This study aims at identifying transcriptional targets of FruitlessBM (FruBM), which represents the major isoform of male-specific FruM transcription factors that induce neural sexual dimorphisms. A promoter of the axon-guidance factor gene robo1 carries the 16-bp palindrome motif Pal1, to which FruM binds. Our genome-wide search for Pal1-homologous sequences yielded ~200 candidate genes. Among these, CG17716 potentially encodes a transmembrane protein with extracellular immunoglobulin (Ig)-like domains similar to Robo1. Indeed, FruBM overexpression reduced CG17716 mRNA and protein expression. In the fru-expressing mAL neuron cluster exhibiting sexual dimorphism, we found that CG17716 knockdown in female neurons completely transformed all neurites to the male-type. Conversely, CG17716 overexpression suppressed male-specific midline crossing of fru-expressing sensory axons. We renamed CG17716 teiresias (tei) based on this feminizing function. We hypothesize that Tei interacts with other Ig superfamily transmembrane proteins, including Robo1, to feminize the neurite patterns in females, whereas FruBM represses tei transcription in males.

Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards

BackgroundMalaria transmission from humans to Anopheles mosquitoes requires the presence of gametocytes in human peripheral circulation, and the dynamics of transmission are determined largely by the density and sex ratio of the gametocytes. Molecular methods are thus employed to measure gametocyte densities, particularly when assessing transmission epidemiology and the efficacy of transmission-blocking interventions. However, accurate quantification of male and female gametocytes with molecular methods requires pure male and female gametocytes as reference standards, which are not widely available.MethodsqRT-PCR assays were used to quantify levels of sex-specific mRNA transcripts in Plasmodium falciparum female and male gametocytes (pfs25 and pfMGET, respectively) using synthetic complimentary RNA standards and in vitro cultured gametocytes. Assays were validated and assay performance was investigated in blood samples of clinical trial participants using these standards and compared to absolute quantification by droplet digital PCR (ddPCR).ResultsThe number of transcript copies per gametocyte were determined to be 279.3 (95% CI 253.5–307.6) for the female-specific transcript pfs25, and 12.5 (95% CI 10.6–14.9) for the male-specific transcript pfMGET. These numbers can be used to convert from transcript copies/mL to gametocyte/mL. The reportable range was determined to be 5.71 × 106 to 5.71 female gametocytes/mL for pfs25, and 1.73 × 107 to 1.73 × 101 male gametocytes/mL for pfMGET. The limit of detection was 3.9 (95% CI 2.5–8.2) female gametocytes/mL for pfs25, and 26.9 (95% CI 19.3–51.7) male gametocytes/mL for PfMGET. Both assays showed minimal intra-assay and inter-assay variability with coefficient of variation < 3%. No cross-reactivity was observed in both assays in uninfected human blood samples. Comparison of results from ddPCR to qRT-PCR assays on clinical blood samples indicated a high-level agreement (ICC = 0.998 for pfs25 and 0.995 for pfMGET).ConclusionsThis study reports the validation of qRT-PCR assays that are able to accurately quantify female and male P. falciparum gametocytes at sub-microscopic densities. The assays showed excellent reproducibility, sensitivity, precision, specificity, and accuracy. The methodology will enable the estimation of gametocyte density in the absence of pure female and male gametocyte standards, and will facilitate clinical trials and epidemiological studies.

Transcription of the Sox30 Gene Is Positively Regulated by Dmrt1 in Nile Tilapia.

The Sox family member Sox30 is highly expressed in the testis of several vertebrate species and has been shown to play key roles in spermiogenesis. However, its transcription regulation remains unclear. Here, we analyzed the Sox30 promoter from the teleost fish Nile tilapia (Oreochromis niloticus) and predicted a putative cis-regulatory element (CRE) for doublesex and mab-3 related transcription factor 1 (Dmrt1), a male-specific transcription factor involved in male sex differentiation. Transcriptional profiling revealed that Sox30 and Dmrt1 similarly exhibited a high expression in tilapia testes from 90 days after hatching (dah) to 300 dah, and the transcription of the Sox30 gene was reduced about one-fold in the testes of male tilapia with Dmrt1 knockdown. Further dual-luciferase reporter assay confirmed that Dmrt1 overexpression significantly promoted transcriptional activity of the Sox30 promoter and this promotion was decreased following the mutation of putative CRE for Dmrt1 within the Sox30 promoter. Chromatin immunoprecipitation-based PCR (ChIP-PCR) and electrophoretic mobility shift assay (EMSA) demonstrated that Dmrt1 directly binds to putative CRE within the Sox30 promoter. These results together indicate that Dmrt1 positively regulates the transcription of the tilapia Sox30 gene by directly binding to specific CRE within the Sox30 promoter.

Mutation of doublesex induces sex-specific sterility of the diamondback moth Plutella xylostella

Doublesex (dsx)the downstream gene in the insect sex determination pathway, plays a critical role in sexual differentiation and development. The functions of dsx have been characterized in several model insect species. However, the molecular mechanism and functions of sex determination of dsx in Plutella xylostella, an agricultural pest, are still unknown. In present study, we identified a male-specific and three female-specific Pxdsx transcripts in P. xylostella. Phylogenetic analyses and multiple sequence alignment revealed that Pxdsx is highly conserved in lepidopterans. The CRISPR/Cas9 technology was used to induce mutations in the male-specific isoform, the female-specific isoform, and common regions of Pxdsx. Disruptions of Pxdsx sex-specific isoforms caused sex-specific defects in external genitals and partial sexual reversal. In addition, we found that female specific transcripts were detected in PxdsxM male mutants and male-specific transcripts were detected in PxdsxF female mutants. Mutations also caused changes in expression of several sex-biased genes and induced sex-specific sterility. This study demonstrates that Pxdsx plays a key role in sex determination of P. xylostella and suggests novel genetic control approaches for the management of P. xylostella.

Identification and characterization of doublesex from the pumpkin fruit fly, Bactrocera tau (Diptera: Tephritidae)

The sex determination cascades in insects are diversified at the top of the cascade, where different primary molecular signals are employed, while at the bottom of the cascades, particularly the doublesex genes, are highly conserved. Here, we identified the doublesex ortholog (Btau-dsx) of Bactrocera tau, a pumpkin fruit fly, and found that Btau-dsx is composed of six exons and five introns with an additional short "m" exon located in the second intron. Btau-dsx is different from its orthologs in most dipteran insects: Its pre-mRNA is sex-specifically spliced to yield three (two male and one female) instead of two transcript variants. The two deduced proteins produced by the male-specific transcripts are a functional (Btau-DSXM1) and a truncated (Btau-DSXM2) protein, while the female-specific transcript produces the functional Btau-DSXF protein. These three proteins contain all conserved domains except Btau-DSXM2 which has no OD2 domain. The female-specific transcript is detected in both fertilized and unfertilized eggs and in both somatic and germ cells of the adult females, while the male-specific transcript is detected only in fertilized eggs and in the abdominal tissues and testes of adult males. The presence of the Btau-dsxM1 transcript in fertilized eggs at the early syncytium stage suggests that in XY embryos, the Y-linked M factor gene may function quite soon after fertilization to alter the splicing pattern of Btau-dsx pre-mRNA from the female-specific to the male-specific mode. Injection of Btau-dsxF dsRNA into recently emerging females can reduce the expression of vitellogenin (Btau-Vg) and causes some defects in the ovaries, indicating that Btau-dsxF works upstream of Btau-Vg.

Distinct core promoter codes drive transcription initiation at key developmental transitions in a marine chordate

BackgroundDevelopment is largely driven by transitions between transcriptional programs. The initiation of transcription at appropriate sites in the genome is a key component of this and yet few rules governing selection are known. Here, we used cap analysis of gene expression (CAGE) to generate bp-resolution maps of transcription start sites (TSSs) across the genome of Oikopleura dioica, a member of the closest living relatives to vertebrates.ResultsOur TSS maps revealed promoter features in common with vertebrates, as well as striking differences, and uncovered key roles for core promoter elements in the regulation of development. During spermatogenesis there is a genome-wide shift in mode of transcription initiation characterized by a novel core promoter element. This element was associated with > 70% of male-specific transcription, including the use of cryptic internal promoters within operons. In many cases this led to the exclusion of trans-splice sites, revealing a novel mechanism for regulating which mRNAs receive the spliced leader. Binding of the cell cycle regulator, E2F1, is enriched at the TSS of maternal genes in endocycling nurse nuclei. In addition, maternal promoters lack the TATA-like element found in zebrafish and have broad, rather than sharp, architectures with ordered nucleosomes. Promoters of ribosomal protein genes lack the highly conserved TCT initiator. We also report an association between DNA methylation on transcribed gene bodies and the TATA-box.ConclusionsOur results reveal that distinct functional promoter classes and overlapping promoter codes are present in protochordates like in vertebrates, but show extraordinary lineage-specific innovations. Furthermore, we uncover a genome-wide, developmental stage-specific shift in the mode of TSS selection. Our results provide a rich resource for the study of promoter structure and evolution in Metazoa.

Ecdysone signaling regulates specification of neurons with a male-specific neurite in Drosophila.

ABSTRACTSome mAL neurons in the male brain form the ipsilateral neurite (ILN[+]) in a manner dependent on FruBM, a male-specific transcription factor. FruBM represses robo1 transcription, allowing the ILN to form. We found that the proportion of ILN[+]-mALs in all observed single cell clones dropped from ∼90% to ∼30% by changing the heat-shock timing for clone induction from 4-5 days after egg laying (AEL) to 6-7 days AEL, suggesting that the ILN[+]-mALs are produced predominantly by young neuroblasts. Upon EcR-A knockdown, ILN[+]-mALs were produced at a high rate (∼60%), even when heat shocked at 6-7 days AEL, yet EcR-B1 knockdown reduced the proportion of ILN[+]-mALs to ∼30%. Immunoprecipitation assays in S2 cells demonstrated that EcR-A and EcR-B1 form a complex with FruBM. robo1 reporter transcription was repressed by FruBM and ecdysone counteracted FruBM. We suggest that ecdysone signaling modulates the FruBM action to produce an appropriate number of male-type neurons.

Isolation and Molecular Characterization of the Transformer Gene From Bactrocera cucurbitae (Diptera: Tephritidae).

transformer (tra) is a switch gene of sex determination in many insects, particularly in Dipterans. However, the sex determination pathway in Bactrocera cucurbitae (Coquillett), a very destructive pest on earth, remains largely uncharacterized. In this study, we have isolated and characterized one female-specific and two male-specific transcripts of the tra gene (Bcutra) of B. cucurbitae. The genomic structure of Bcutra has been determined and the presence of multiple conserved Transformer (TRA)/TRA-2 binding sites in Bcutra has been found. BcuTRA is highly conservative with its homologues in other tephritid fruit flies. Gene expression analysis of Bcutra at different developmental stages demonstrates that the female transcript of Bcutra appears earlier than the male counterparts, indicating that the maternal TRA is inherited in eggs and might play a role in the regulation of TRA expression. The conservation of protein sequence and sex-specific splicing of Bcutra and its expression patterns during development suggest that Bcutra is probably the master gene of sex determination of B. cucurbitae. Isolation of Bcutra will facilitate the development of a genetic sexing strain for its biological control.

Male-Specific Transcription Factor Occupancy Alone Does Not Account for Differential Methylation at Imprinted Genes in the mouse Germ Cell Lineage.

Genomic imprinting is an epigenetic mechanism that affects a subset of mammalian genes, resulting in monoallelic expression depending on the parental origin of the alleles. Imprinted regions contain regulatory elements that are methylated in the gametes in a sex-specific manner (differentially methylated regions; DMRs). DMRs are present at nonimprinted loci as well, but whereas most regions are equalized after fertilization, methylation at imprinted regions maintains asymmetry. We tested the hypothesis that paternally unmethylated DMRs are occupied by transcription factors (TFs) present during male gametogenesis. Meta-analysis of mouse RNA data to identify DNA-binding proteins expressed in male gametes and motif enrichment analysis of active promoters yielded a list of candidate TFs. We then asked whether imprinted or nonimprinted paternally unmethylated DMRs harbored motifs for these TFs, and found many shared motifs between the two groups. However, DMRs that are methylated in the male germ cells also share motifs with DMRs that remain unmethylated. There are recognition sequences exclusive to the unmethylated DMRs, whether imprinted or not, that correspond with cell-cycle regulators, such as p53. Thus, at least with the current available data, our results indicate a complex scenario in which TF occupancy alone is not likely to play a role in protecting unmethylated DMRs, at least during male gametogenesis. Rather, the epigenetic features of DMRs, regulatory sequences other than DMRs, and the role of DNA-binding proteins capable of endowing sequence specificity to DNA-methylating enzymes are feasible mechanisms and further investigation is needed to answer this question.