RESUMO
Meiotic drive loci distort the normally equal segregation of alleles, which benefits their own transmission even in the face of severe fitness costs to their host organism. However, relatively little is known about the molecular identity of meiotic drivers, their strategies of action, and mechanisms that can suppress their activity. Here, we present data from the fruitfly Drosophila simulans that address these questions. We show that a family of de novo, protamine-derived X-linked selfish genes (the Dox gene family) is silenced by a pair of newly emerged hairpin RNA (hpRNA) small interfering RNA (siRNA)-class loci, Nmy and Tmy. In the w[XD1] genetic background, knockout of nmy derepresses Dox and MDox in testes and depletes male progeny, whereas knockout of tmy causes misexpression of PDox genes and renders males sterile. Importantly, genetic interactions between nmy and tmy mutant alleles reveal that Tmy also specifically maintains male progeny for normal sex ratio. We show the Dox loci are functionally polymorphic within D. simulans, such that both nmy-associated sex ratio bias and tmy-associated sterility can be rescued by wild-type X chromosomes bearing natural deletions in different Dox family genes. Finally, using tagged transgenes of Dox and PDox2, we provide the first experimental evidence Dox family genes encode proteins that are strongly derepressed in cognate hpRNA mutants. Altogether, these studies support a model in which protamine-derived drivers and hpRNA suppressors drive repeated cycles of sex chromosome conflict and resolution that shape genome evolution and the genetic control of male gametogenesis.
Assuntos
Drosophila simulans , Cromossomos Sexuais , Animais , Masculino , Drosophila simulans/genética , Cromossomos Sexuais/genética , Drosophila/genética , Cromossomo X , RNA Interferente Pequeno/genética , Razão de Masculinidade , Meiose/genéticaRESUMO
Segregation Distorter (SD) is a male meiotic drive system in Drosophila melanogaster. Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD+ chromosome. It is well established that distortion results from an interaction between Sd, the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp, on the SD+ chromosome. However, the molecular and cellular mechanisms leading to post-meiotic SD+ sperm elimination remain unclear. Here we show that SD/SD+ males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD+ spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD+ genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. Our analyses of different SD+ chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. We propose that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization.
Assuntos
DNA Satélite/genética , Proteínas de Drosophila/genética , Proteínas Ativadoras de GTPase/genética , Espermatogênese/genética , Animais , Núcleo Celular/metabolismo , Cromatina/genética , Mapeamento Cromossômico , Segregação de Cromossomos , Cromossomos/genética , DNA Satélite/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Ativadoras de GTPase/metabolismo , Genótipo , Masculino , Meiose , Mutação , Fenótipo , Espermátides/metabolismo , Espermatozoides/metabolismoRESUMO
Sex chromosome meiotic drive, the non-Mendelian transmission of sex chromosomes, is the expression of an intragenomic conflict that can have extreme evolutionary consequences. However, the molecular bases of such conflicts remain poorly understood. Here, we show that a young and rapidly evolving X-linked heterochromatin protein 1 (HP1) gene, HP1D2, plays a key role in the classical Paris sex-ratio (SR) meiotic drive occurring in Drosophila simulans Driver HP1D2 alleles prevent the segregation of the Y chromatids during meiosis II, causing female-biased sex ratio in progeny. HP1D2 accumulates on the heterochromatic Y chromosome in male germ cells, strongly suggesting that it controls the segregation of sister chromatids through heterochromatin modification. We show that Paris SR drive is a consequence of dysfunctional HP1D2 alleles that fail to prepare the Y chromosome for meiosis, thus providing evidence that the rapid evolution of genes controlling the heterochromatin structure can be a significant source of intragenomic conflicts.
Assuntos
Evolução Molecular , Heterocromatina/metabolismo , Meiose/genética , Cromossomo Y , Animais , Drosophila simulans/classificação , Drosophila simulans/genética , FilogeniaRESUMO
In Drosophila, telomere-capping proteins have the remarkable capacity to recognize chromosome ends in a sequence-independent manner. This epigenetic protection is essential to prevent catastrophic ligations of chromosome extremities. Interestingly, capping proteins occupy a large telomere chromatin domain of several kilobases; however, the functional relevance of this to end protection is unknown. Here, we investigate the role of the large capping domain by manipulating HOAP (encoded by caravaggio) capping-protein expression in the male germ cells, where telomere protection can be challenged without compromising viability. We show that the exhaustion of HOAP results in a dramatic reduction of other capping proteins at telomeres, including K81 [encoded by ms(3)K81], which is essential for male fertility. Strikingly however, we demonstrate that, although capping complexes are barely detected in HOAP-depleted male germ cells, telomere protection and male fertility are not dramatically affected. Our study thus demonstrates that efficient protection of Drosophila telomeres can be achieved with surprisingly low amounts of capping complexes. We propose that these complexes prevent fusions by acting at the very extremity of chromosomes, reminiscent of the protection conferred by extremely short telomeric arrays in yeast or mammalian systems.
Assuntos
Proteínas de Drosophila/genética , Espermatogênese/genética , Telômero/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Masculino , Telômero/metabolismoRESUMO
In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.
Assuntos
Centrômero/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Histonas/metabolismo , Padrões de Herança/genética , Espermatozoides/metabolismo , Animais , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário/genética , Fertilização , Fase G2/genética , Proteínas de Fluorescência Verde/metabolismo , Cinetocoros/metabolismo , Masculino , Proteínas Recombinantes de Fusão/metabolismo , Espermatogênese/genéticaRESUMO
Intra-genomic conflict driven by selfish chromosomes is a powerful force that shapes the evolution of genomes and species. In the male germline, many selfish chromosomes bias transmission in their own favor by eliminating spermatids bearing the competing homologous chromosomes. However, the mechanisms of targeted gamete elimination remain mysterious. Here, we show that Overdrive (Ovd), a gene required for both segregation distortion and male sterility in Drosophila pseudoobscura hybrids, is broadly conserved in Dipteran insects but dispensable for viability and fertility. In D. melanogaster, Ovd is required for targeted Responder spermatid elimination after the histone-to-protamine transition in the classical Segregation Distorter system. We propose that Ovd functions as a general spermatid quality checkpoint that is hijacked by independent selfish chromosomes to eliminate competing gametes.
RESUMO
Circadian rhythms are generated by well-conserved interlocked transcriptional feedback loops in animals. In Drosophila, the dimeric transcription factor CLOCK/CYCLE (CLK/CYC) promotes period (per), timeless (tim), vrille (vri), and PAR-domain protein 1 (Pdp1) transcription. PER and TIM negatively feed back on CLK/CYC transcriptional activity, whereas VRI and PDP1 negatively and positively regulate Clk transcription, respectively. Here, we show that the α isoform of the Drosophila FOS homolog KAYAK (KAY) is required for normal circadian behavior. KAY-α downregulation in circadian pacemaker neurons increases period length by 1.5 h. This behavioral phenotype is correlated with decreased expression of several circadian proteins. The strongest effects are on CLK and the neuropeptide PIGMENT DISPERSING FACTOR, which are both under VRI and PDP1 control. Consistently, KAY-α can bind to VRI and inhibit its interaction with the Clk promoter. Interestingly, KAY-α can also repress CLK activity. Hence, in flies with low KAY-α levels, CLK derepression would partially compensate for increased VRI repression, thus attenuating the consequences of KAY-α downregulation on CLK targets. We propose that the double role of KAY-α in the two transcriptional loops controlling Drosophila circadian behavior brings precision and stability to their oscillations.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Retroalimentação Fisiológica/fisiologia , Neurônios/fisiologia , Transcrição Gênica/fisiologia , Animais , Animais Geneticamente Modificados/fisiologia , Relógios Biológicos/genética , Células Cultivadas , DNA/genética , Proteínas de Drosophila/genética , Células HEK293 , Humanos , Imuno-Histoquímica , Atividade Motora/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/fisiologia , Plasmídeos/genética , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/genética , TransfecçãoRESUMO
The global replacement of histones with protamines in sperm chromatin is widespread in animals, including insects, but its actual function remains enigmatic. We show that in the Drosophila paternal effect mutant paternal loss (pal), sperm chromatin retains germline histones H3 and H4 genome wide without impairing sperm viability. However, after fertilization, pal sperm chromosomes are targeted by the egg chromosomal passenger complex and engage into a catastrophic premature division in synchrony with female meiosis II. We show that pal encodes a rapidly evolving transition protein specifically required for the eviction of (H3-H4)2 tetramers from spermatid DNA after the removal of H2A-H2B dimers. Our study thus reveals an unsuspected role of histone eviction from insect sperm chromatin: safeguarding the integrity of the male pronucleus during female meiosis.
Assuntos
Amidina-Liases , Cromatina , Proteínas de Drosophila , Drosophila melanogaster , Fertilização , Histonas , Herança Paterna , Espermatozoides , Animais , Feminino , Masculino , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Espermatozoides/metabolismo , Amidina-Liases/genética , Amidina-Liases/metabolismo , Empacotamento do DNARESUMO
Circadian pacemakers are essential to synchronize animal physiology and behavior with the dayrationight cycle. They are self-sustained, but the phase of their oscillations is determined by environmental cues, particularly light intensity and temperature cycles. In Drosophila, light is primarily detected by a dedicated blue-light photoreceptor: CRYPTOCHROME (CRY). Upon light activation, CRY binds to the pacemaker protein TIMELESS (TIM) and triggers its proteasomal degradation, thus resetting the circadian pacemaker. To understand further the CRY input pathway, we conducted a misexpression screen under constant light based on the observation that flies with a disruption in the CRY input pathway remain robustly rhythmic instead of becoming behaviorally arrhythmic. We report the identification of more than 20 potential regulators of CRY-dependent light responses. We demonstrate that one of them, the chromatin-remodeling enzyme KISMET (KIS), is necessary for normal circadian photoresponses, but does not affect the circadian pacemaker. KIS genetically interacts with CRY and functions in PDF-negative circadian neurons, which play an important role in circadian light responses. It also affects daily CRY-dependent TIM oscillations in a peripheral tissue: the eyes. We therefore conclude that KIS is a key transcriptional regulator of genes that function in the CRY signaling cascade, and thus it plays an important role in the synchronization of circadian rhythms with the dayrationight cycle.
Assuntos
Ritmo Circadiano/genética , Ritmo Circadiano/efeitos da radiação , DNA Helicases/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Testes Genéticos , Proteínas de Homeodomínio/genética , Luz , Animais , Comportamento Animal/efeitos da radiação , Criptocromos/genética , Criptocromos/metabolismo , DNA Helicases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Genes de Insetos/genética , Proteínas de Homeodomínio/metabolismo , Neurônios/metabolismo , Neurônios/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , RNA de Cadeia Dupla/metabolismoRESUMO
Circadian clocks synchronize the physiology and behavior of most animals with the day to night cycle. A fundamental property of the molecular pacemakers generating circadian rhythms is their self-sustained nature: they keep oscillating even under constant conditions, with a period close to, but not exactly, 24 h. However, circadian pacemakers have to be sensitive to environmental cues to be beneficial. They need to be reset every day to keep a proper phase relationship with the day to night cycle, and they have to be able to adjust to seasonal changes in day length and temperature. Here, we review our current knowledge of the molecular and neural mechanisms contributing to the plasticity of Drosophila circadian rhythms, which are proving to be remarkably sophisticated and complex.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Sinais (Psicologia) , Drosophila/fisiologia , Meio Ambiente , Plasticidade Neuronal/fisiologia , Ciclos de Atividade/fisiologia , Animais , Comportamento Animal/fisiologia , Regulação da Expressão Gênica , Luz , Atividade Motora/fisiologia , Rede Nervosa , Temperatura , TransgenesRESUMO
Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. Here, we reveal that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, we generated D. simulans null mutants of Dcr-2 and AGO2. Strikingly, these core RNAi knockouts massively derepress Dox and MDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, our data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species.
Assuntos
Células Germinativas/metabolismo , Meiose/genética , Interferência de RNA/fisiologia , Espermatozoides/metabolismo , Animais , Proteínas Argonautas/genética , Drosophila , Proteínas de Drosophila/genética , Evolução Molecular , Masculino , RNA Helicases/genética , RNA Interferente Pequeno/genética , Ribonuclease III/genéticaRESUMO
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.
Assuntos
Cromatina/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fertilização/fisiologia , Proteínas de Membrana/metabolismo , Óvulo/fisiologia , Espermatozoides/fisiologia , Tiorredoxinas/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Proteínas de Membrana/genética , Tiorredoxinas/genéticaRESUMO
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 cellular and nuclear processes, such as sperm-egg cellular interactions, sperm chromatin remodelling, centrosome formation or pronuclear migration. In comparison with other aspects of development, the exploration of animal fertilization at the functional level has remained so far relatively limited, even in classical model organisms. Here, we have reviewed our current knowledge of fertilization in Drosophila melanogaster, with a special emphasis on the genes involved in the complex transformation of the fertilizing sperm nucleus into a replicated set of paternal chromosomes.
Assuntos
Drosophila/genética , Fertilização/fisiologia , Animais , Divisão Celular , Núcleo Celular , Feminino , Masculino , Mutação , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologia , Zigoto/metabolismoRESUMO
A negative transcriptional feedback loop generates circadian rhythms in Drosophila. PERIOD (PER) is a critical state-variable in this mechanism, and its abundance is tightly regulated. We found that the Drosophila homolog of ATAXIN-2 (ATX2)--an RNA-binding protein implicated in human neurodegenerative diseases--was required for circadian locomotor behavior. ATX2 was necessary for PER accumulation in circadian pacemaker neurons and thus determined period length of circadian behavior. ATX2 was required for the function of TWENTY-FOUR (TYF), a crucial activator of PER translation. ATX2 formed a complex with TYF and promoted its interaction with polyadenylate-binding protein (PABP). Our work uncovers a role for ATX2 in circadian timing and reveals that this protein functions as an activator of PER translation in circadian neurons.
Assuntos
Ritmo Circadiano , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Proteínas Circadianas Period/biossíntese , Animais , Ataxinas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a Poli(A)/metabolismo , Biossíntese de Proteínas , Interferência de RNARESUMO
Comparative genome analysis has allowed the identification of various mechanisms involved in gene birth. However, understanding the evolutionary forces driving new gene origination still represents a major challenge. In particular, an intriguing and not yet fully understood trend has emerged from the study of new genes: many of them show a testis-specific expression pattern, which has remained poorly understood. Here we review the case of such a new gene, which involves a telomere-capping gene family in Drosophila. hiphop and its testis-specific paralog K81 are critical for the protection of chromosome ends in somatic cells and male gametes, respectively. Two independent functional studies recently proposed that these genes evolved under a reproductive-subfunctionalization regime. The 2011 release of new Drosophila genome sequences from the melanogaster group of species allowed us to deepen our phylogenetic analysis of the hiphop/K81 family. This work reveals an unsuspected dynamic of gene birth and death within the group, with recurrent duplication events through retroposition mechanisms. Finally, we discuss the plausibility of different evolutionary scenarios that could explain the diversification of this gene family.
RESUMO
A critical function of telomeres is to prevent the ligation of chromosome ends by DNA repair enzymes. In most eukaryotes, telomeric DNA consists in large arrays of G-rich tandem repeats that are recognized by DNA binding capping proteins. Drosophila telomeres are unusual as they lack short tandem repeats. However, Drosophila capping proteins can bind chromosome extremities in a DNA sequence-independent manner. This epigenetic protection of fly telomeres has been essentially studied in somatic cells where capping proteins such as HOAP or HP1 are essential in preventing chromosome end-to-end fusions. HipHop and K81 are two recently identified paralogous capping proteins with complementary expression patterns. While HipHop is involved in telomere capping in somatic cells, K81 has specialized in the protection of telomeres in post-meiotic male germ cells. Remarkably, K81 is required for the stabilization of HOAP and HP1 at telomeres during the massive paternal chromatin remodeling that occurs during spermiogenesis and at fertilization. We thus propose that the maintenance of capping proteins at Drosophila sperm telomeres is crucial for the transmission of telomere identity to the diploid zygote. :
Assuntos
Montagem e Desmontagem da Cromatina/genética , Epigênese Genética/genética , Fertilização/genética , Espermatogênese/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fertilização/fisiologia , Masculino , Espermatogênese/fisiologia , Telômero/genética , Proteínas de Ligação a Telômeros/genéticaRESUMO
BACKGROUND: A critical function of telomeres is to prevent fusion of chromosome ends by the DNA repair machinery. In Drosophila somatic cells, assembly of the protecting capping complex at telomeres notably involves the recruitment of HOAP, HP1, and their recently identified partner, HipHop. We previously showed that the hiphop gene was duplicated before the radiation of the melanogaster subgroup of species, giving birth to K81, a unique paternal effect gene specifically expressed in the male germline. RESULTS: Here we show that K81 specifically associates with telomeres during spermiogenesis, along with HOAP and HP1, and is retained on paternal chromosomes until zygote formation. In K81 mutant testes, capping proteins are not maintained at telomeres in differentiating spermatids, resulting in the transmission of uncapped paternal chromosomes that fail to properly divide during the first zygotic mitosis. Despite the apparent similar capping roles of K81 and HipHop in their respective domain of expression, we demonstrate by in vivo reciprocal complementation analyses that they are not interchangeable. Strikingly, HipHop appeared to be unable to maintain capping proteins at telomeres during the global chromatin remodeling of spermatid nuclei. CONCLUSIONS: Our data demonstrate that K81 is essential for the maintenance of capping proteins at telomeres in postmeiotic male germ cells. In species of the melanogaster subgroup, HipHop and K81 have not only acquired complementary expression domains, they have also functionally diverged following the gene duplication event. We propose that K81 specialized in the maintenance of telomere protection in the highly peculiar chromatin environment of differentiating male gametes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Espermatozoides/fisiologia , Telômero/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Epigênese Genética , Feminino , Masculino , Família Multigênica , Filogenia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, we identified novel genes regulated by dRFX in Drosophila. RESULTS: We first demonstrate that a subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. We then designed an X-box consensus sequence and carried out a genome wide computer screen to identify novel genes under RFX control. We found 412 genes that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. We analyzed 25 of these 83 genes, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons. CONCLUSION: Our X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. We also established a highly valuable Drosophila cilia and basal body dataset. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-Biedl syndrome.
Assuntos
Proteínas de Ligação a DNA/genética , Drosophila/genética , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Motivos de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans , Sequência Conservada , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Genes Reporter , Genômica/métodos , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição de Fator Regulador X , Especificidade da EspécieRESUMO
Ciliated neurons play an important role in sensory perception in many animals. Modified cilia at dendrite endings serve as sites of sensory signal capture and transduction. We describe Drosophila mutations that affect the transcription factor RFX and genetic rescue experiments that demonstrate its central role in sensory cilium differentiation. Rfx mutant flies show defects in chemosensory and mechanosensory behaviors but have normal phototaxis, consistent with Rfx expression in ciliated sensory neurons and neuronal precursors but not in photoreceptors. The mutant behavioral phenotypes are correlated with abnormal function and structure of neuronal cilia, as shown by the loss of sensory transduction and by defects in ciliary morphology and ultrastructure. These results identify Rfx as an essential regulator of ciliated sensory neuron differentiation in Drosophila.