[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Nov 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: 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
and its testis-specific paralog
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
genome sequences from the
group of species allowed us to deepen our phylogenetic analysis of the
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.
[Show abstract][Hide abstract] ABSTRACT: 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. :
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
Full-text · Article · Dec 2010 · Current biology: CB
[Show abstract][Hide abstract] ABSTRACT: cry overexpression restores constant light arrhythmicity in flies expressing kis dsRNAs. (A) Actograms showing the locomotor activity of tim-GAL4 VDRC46685/+ (tg4 VDRC46685;+); and tim-GAL4 VDRC46685/UAS-cry flies (tg4 VDRC46685;cry) under LL conditions. Both genotypes are homozygous for ls-tim (n = 16 flies for each genotype). (B) Percentage of LL rhythmicity for the same genotypes.
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[Show abstract][Hide abstract] ABSTRACT: KIS protein levels are severely reduced in circadian neurons expressing kis dsRNAs. (A) KIS protein levels in large and small LNvs and in LNds measured by immunostaining. The histogram represents the normalized KIS fluorescence signal (see Materials and Methods for details), measured in control flies (pdf-GAL4 UAS-dcr2/+ [pg4 dcr2/+] and tim-GAL4 UAS-dcr2; R32 [tg4 dcr2/+]) and flies expressing kis dsRNAs in all circadian neurons (tim-GAL4 UAS-dcr2/VDRC46685; R32 [tg4 dcr2/VDRC46685]) or only in PDF positive neurons (pdf-GAL4 UAS-dcr2/VDRC46685 [pg4 dcr2/VDRC46685]). KIS expression is reduced by about 85–90% in both mutant genotypes, and in all cell types surveyed. When driving kis dsRNAs with pdf-GAL4, we used an anti-PDF antibody to identify the PDF positive LNvs. With tim-GAL4, we used flies carrying one copy of the R32 lacZ insertion trap  and identified clock neurons with an anti-βGAL antibody. βGAL staining was weak in Dorsal Neurons, but a few DN1s could nevertheless be identified in tg4 dcr2/VDRC46685 brains. KIS expression was also severely reduced in these neurons (data not shown). Error bars represent standard deviations. (B) Histogram showing the percentage of rhythmicity in constant light for control flies (pdf-GAL4 UAS-dcr2/+ [pg4 dcr2/+] and tim-GAL4 UAS-dcr2/+ [tg4 dcr2/+]) and flies expressing kis dsRNAs in all circadian neurons (tim-GAL4 UAS-dcr2/VDRC46685 [tg4 dcr2/VDRC46685]; ls-tim homozygotes) or only in PDF positive neurons (pdf-GAL4 UAS-dcr2/VDRC46685 [pg4 dcr2/VDRC46685]; ls-tim homozygotes) (n = 16 flies for each genotype). As also shown on Figure 5 with the NIG-Fly line, driving the VDRC kis dsRNAs only in PDF positive LNvs does not induce LL rhythmicity, even though as shown in (A) KIS expression is as efficiently repressed as with tim-GAL4. AR: complete arrhythmicity.
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[Show abstract][Hide abstract] ABSTRACT: Behavior of EP(2)2356 crossed to different GAL4 drivers and GAL80 repressor transgenes under constant light (200 lux, unless otherwise indicted) (AR = arrhythmicity).
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[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the
immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality
and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the
embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast
to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia
during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates
the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion,
RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.
Full-text · Article · Jun 2004 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: 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.