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 displace for Molecular Parasitology and Infection and Immunity. Glasgow G12 8TA. UK
Subject areas: Development. Molecular biology. Genetics. Genome studies
© 2007 Laurençon et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution authorise () which permits unrestricted use distribution and reproduction in any medium provided the original bring home the bacon is properly cited.
and walk. Using the tremendous advantages of comparative genomics in closely related species we identified novel genes regulated by dRFX in
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 open 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 create expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of
cilia and basal body dataset. These results show 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.
Eukaryotic cilia and flagella are show in many types of tissues and organisms and are important for sensory functions cell motility molecular transport and several developmental processes such as the establishment of left-right asymmetry in vertebrates []. Several human diseases are known to prove from defects in ciliary assembly or answer and have recently been designated as ciliopathies []. Cilia are well-defined structures consisting of a microtubular axoneme composed of specific proteins that are assembled dynamically in a strict stereotypical copy (for reviews see [,]). Ciliary assembly depends on intraflagellar transport (IFT) a dynamic process highly conserved in organisms ranging from the color algae
to mammals (reviewed in [,,]). Several studies in various organisms have been instrumental in the identification of genes involved in the assembly and answer of the cilium. The proteomic analysis of detergent-extracted ciliary axonemes from cultured human epithelial cells identified 214 proteins []. More recently a biochemical fractionation of
flagella led to the identification of about 700 proteins of which 360 had high confidence of truly being involved in flagellar composition []. A proteomic analysis of
flagella allowed the identification of 522 proteins []. Two remarkable approaches took advantage of the availability of complete genome sequences to identify genes encoding ciliary and flagellar proteins. By comparing the genomes of ciliated versus non-ciliated organisms. Avidor-Reiss
genes after deflagellation. They identified 220 genes that are induced at least two-fold and therefore are likely to be involved in the assembly or answer of cilia and flagella.
Much less is known about the regulatory pathways that control the expression of ciliary components or enjoin the differentiation of ciliated cells. The transcription factor FoxJ1 appears to govern the differentiation of ciliated cells in vertebrates but so far only one gene has been shown to be directly regulated by FoxJ1 []. The transcription calculate HNF1-β has also been shown to regulate several genes involved in ciliogenesis in the kidney []. Most importantly regulatory calculate X (RFX) transcription factors play a key role in regulating genes involved in ciliogenesis. RFX transcription factors are conserved in a wide be of species including
and mammals. They overlap a characteristic DNA-binding domain of the winged-helix DNA binding family and attach to an X-box motif an imperfect inverted repeat with variable spacing between the repeats [,]. Whereas only one
is expressed in ciliated cells and is necessary for ciliated sensory neuron differentiation: all sensory neurons are present but cilia are missing at the dendritic tips [,]. In mouse we have shown that RFX answer in ciliogenesis is conserved. Indeed.
loss-of-function leads to hydrocephalus with differentiation defects of ciliated ependymal cells of the choroid plexus and subcommisural organ []. Moreover.
mutant mice show insulin secretion failure and impaired glucose tolerance correlated with primary ciliary growth defects on islet cells []. In zebrafish.
the most divergent mammalian member regulates major histocompatibility class II gene expression and mutations in it are responsible for the bare lymphocyte syndrome [].
has been implicated in dorsal patterning of brain development in mice and may participate in circadian rhythm regulation in humans [].
to mammals. X-box promoter motif sequences can guide the search for ciliary genes. Indeed genome wide searches for genes controlled by DAF-19 in
have identified many genes involved in ciliogenesis [,,]. Genomic X-box searches thus comprise a key method to identify genes involved in ciliary development. We show here that ciliogenic RFX regulatory cascades are well conserved between
homologs of genes defective in human Bardet-Biedl syndrome (BBS) a human ciliopathy with complex phenotypes are controlled by dRFX. Moreover by using comparative genomic screens we show that genes under dRFX hold back in
reporter assay studies for three of them confirmed their involvement in ciliary structure or function in
cilia and basal be (DCBB) gene list and highlight several genes as novel candidates for ciliogenesis. Our data are of particular importance for advance genetic and genomic studies in the field of ciliogenesis and consequently for identifying genes involved in human ciliopathies.
Our previous work has shown that RFX transcription factors share a common answer in ciliogenesis in worm and fly [,]. We thus inferred that an identical set of genes would be regulated by DAF-19 in
mutants (Table ). Regulation of gene expression was tested by real-time PCR based on RNA extracted from 40-hour old pupae thoraxes and legs. At this stage dendrites and cilia undergo just differentiated. Moreover the levels of expression of ciliary genes
during pupae development is at a maximum starting at 40 hours after puparium formation (data not shown). As shown in Table. 14 of 19 DAF-19 regulated genes for which a homologous gene can be found in
do not appear to be regulated by dRFX in our assay conditions. However we cannot exclude that these genes are under dRFX regulation in a small subset of ciliated sensory neurons and thus that variations of their expression cannot be detected by real measure RT-PCR of RNA preparations of pupae thoraxes and legs. Remarkably genes that are involved in BBS and conserved in both organisms are regulated by RFX proteins. We quantified the expression of
deficient background. Most of the other genes regulated by dRFX are involved in IFT. This transport is led by two types of molecular motors anterograde kinesins and orb dyneins that carry particles that can be biochemically fractionated.
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http://genomebiology.com/2007/8/9/R195
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