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Target Genes by Comparative Genomics in Drosophila Species Open Access Research2007LaurençonetVolume al. 8, Issue 9, Article R195 Identification of novel regulatory factor X (RFX) target genes by comment comparative genomics in Drosophila species Anne Laurençon*†, Raphaëlle Dubruille*†‡, Evgeni Efimenko§, Guillaume Grenier*†, Ryan Bissett*†¶, Elisabeth Cortier*†, Vivien Rolland*†, Peter Swoboda§ and Bénédicte Durand*† * † Addresses: Université de Lyon, Lyon, F-69003, France. Université Lyon 1, CNRS, UMR5534, Centre de Génétique Moléculaire et Cellulaire, reviews Villeurbanne, F-69622, France. ‡University of Massachusetts Medical School, Department of Neurobiology, Worcester, MA 01605, USA. §Karolinska Institute, Department of Biosciences and Nutrition, Södertörn University College, School of Life Sciences, S-14189 Huddinge, Sweden. ¶University of Glasgow, Glasgow Biomedical Research Centre, Wellcome Centre for Molecular Parasitology and Infection and Immunity, Glasgow G12 8TA, UK. Correspondence: Anne Laurençon. Email: [email protected] Published: 17 September 2007 Received: 23 July 2007 Revised: 14 September 2007 reports Genome Biology 2007, 8:R195 (doi:10.1186/gb-2007-8-9-r195) Accepted: 17 September 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/9/R195 © 2007 Laurençon et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. deposited research Novel<p>AnDrosophila RFX RFX-binding targetspecies genes identified site is shown novel to RFX be conserved target genes in thethat promoter are involveds of ain subset sensory of ciliaryciliogenesis.</p> genes and a subsequent screen for this site in two Abstract 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. research refereed 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 interactions 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 information syndrome. Genome Biology 2007, 8:R195 R195.2 Genome Biology 2007, Volume 8, Issue 9, Article R195 Laurençon et al. http://genomebiology.com/2007/8/9/R195 Background gene in C. elegans, is a key regulator of ciliogenesis [21]. dRfx Eukaryotic cilia and flagella are present in many types of tis- in Drosophila is expressed in ciliated cells and is necessary for sues and organisms and are important for sensory functions, ciliated sensory neuron differentiation: all sensory neurons cell motility, molecular transport, and several developmental are present but cilia are missing at the dendritic tips [22,23]. processes, such as the establishment of left-right asymmetry In mouse, we have shown that RFX function in ciliogenesis is in vertebrates [1-5]. Several human diseases are known to conserved. Indeed, Rfx3 controls the growth of mouse embry- result from defects in ciliary assembly or function and have onic node cilia [24] and Rfx3 loss-of-function leads to hydro- recently been designated as ciliopathies [5]. Cilia are well- cephalus with differentiation defects of ciliated ependymal defined structures consisting of a microtubular axoneme cells of the choroid plexus and subcommisural organ [25]. composed of specific proteins that are assembled dynamically Moreover, Rfx3 mutant mice show insulin secretion failure in a strict stereotypical pattern (for reviews, see [6,7]). Ciliary and impaired glucose tolerance correlated with primary cili- assembly depends on intraflagellar transport (IFT) a dynamic ary growth defects on islet cells [26]. In zebrafish, Rfx2 is process highly conserved in organisms ranging from the expressed specifically in multiciliated cells of the pronephros green algae Chlamydomonas to mammals (reviewed in and loss of Rfx2 leads to cyst formation and loss of multicilia [1,8,9]). Several studies in various organisms have been [27]. The function of the other RFX proteins has yet to be instrumental in the identification of genes involved in the linked to ciliogenesis. Rfx5, the most divergent mammalian assembly and function of the cilium. The proteomic analysis member, regulates major histocompatibility class II gene of detergent-extracted ciliary axonemes from cultured human expression and mutations in it are responsible for the bare epithelial cells identified 214 proteins [10]. More recently, a lymphocyte syndrome [28]. Rfx4 has been implicated in dor- biochemical fractionation of Chlamydomonas reinhardtii sal patterning of brain development in mice and may partici- flagella led to the identification of about 700 proteins, of pate in circadian rhythm regulation in humans [29-32]. which 360 had high confidence of truly being involved in flag- ellar composition [11]. A proteomic analysis of Trypanosoma Because RFX function in ciliogenesis appears conserved from brucei flagella allowed the identification of 522 proteins [12]. C. elegans to mammals, X-box promoter motif sequences can Two remarkable approaches took advantage of the availabil- guide the search for ciliary genes. Indeed, genome wide ity of complete genome sequences to identify genes encoding searches for genes controlled by DAF-19 in C. elegans have ciliary and flagellar proteins. By comparing the genomes of identified many genes involved in ciliogenesis [14,21,33-38]. ciliated versus non-ciliated organisms, Avidor-Reiss et al. Genomic X-box searches thus comprise a key method to iden- [13] and Li et al. [14] selected 187 and 688 genes, respectively, tify genes involved in ciliary development. We show here that that are specific to ciliated organisms. Stolc et al. [15] used ciliogenic RFX regulatory cascades are well conserved microarray hybridization to analyze induction levels of all C. between D. melanogaster and C. elegans and identify a first reinhardtii genes after deflagellation. They identified 220 set of 14 RFX target genes. In particular, we show that all genes that are induced at least two-fold and, therefore, are known Drosophila homologs of genes defective in human likely to be involved in the assembly or function of cilia and Bardet-Biedl syndrome (BBS), a human ciliopathy with com- flagella. plex phenotypes, are controlled by dRFX. Moreover, by using comparative genomic screens we show that genes under Much less is known about the regulatory pathways that con- dRFX control in D. melanogaster share conserved X-boxes trol the expression of ciliary components or direct the differ- with another divergent Drosophila species, D. pseudoob- entiation of ciliated cells. The transcription factor FoxJ1 scura. Applied to the whole genome of both species, our com- appears to govern the differentiation of ciliated cells in verte- parative approach led to the identification of at least 11 novel brates, but so far, only one gene has been shown to be directly RFX target genes. In vivo reporter assay studies for three of regulated by FoxJ1 [16]. The transcription factor HNF1-β has them confirmed their involvement in ciliary structure or func- also been shown to regulate several genes involved in cilio- tion in Drosophila, thus illustrating the accuracy of our genesis in the kidney [17]. Most importantly, regulatory factor screen. In addition, we have established a highly confident X (RFX) transcription factors play a key role in regulating Drosophila cilia and basal body (DCBB) gene list and high- genes involved in ciliogenesis. RFX transcription factors are light several genes as novel candidates for ciliogenesis. Our conserved in a wide range of species, including Saccharomy- data are of particular importance for further genetic and ces cerevisiae, Caenorhabditis elegans, Drosophila mela- genomic studies in the field of ciliogenesis and, consequently, nogaster and mammals. They share a characteristic DNA- for identifying genes involved in human ciliopathies. binding domain of the winged-helix DNA binding family and bind to an X-box motif, an imperfect inverted repeat with var- iable spacing between the repeats [18,19]. Whereas only one Results Rfx gene is described in
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