Identification of novel regulatory factor X (RFX) target genes by comparative genomics in <it>Drosophila </it>species

gb-2007-8-9-r195 GBJ Research Identification of novel regulatory factor X (RFX) target genes by comparative genomics in <it>Drosophila </it>species Laurençon Anne laurencon@cgmc.univ-lyon1.fr Dubruille Raphaëlle Raphaelle.Dubruille@umassmed.edu Efimenko Evgeni evgeni.efimenko@biosci.ki.se Grenier Guillaume grenier@cgmc.univ-lyon1.fr Bissett Ryan r.bissett.1@research.gla.ac.uk Cortier Elisabeth cortier@cgmc.univ-lyon1.fr Rolland Vivien v99rolland@hotmail.com Swoboda Peter peter.swoboda@biosci.ki.se Durand Bénédicte benedicte.durand@univ-lyon1.fr

Université de Lyon, Lyon, F-69003, France

Université Lyon 1, CNRS, UMR5534, Centre de Génétique Moléculaire et Cellulaire, 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

Genome Biology 1465-6906 2007 8 9 R195 http://genomebiology.com/2007/8/9/R195 17875208 10.1186/gb-2007-8-9-r195 23 7 2007 14 9 2007 17 9 2007 17 09 2007 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.

Novel RFX target genes

An RFX-binding site is shown to be conserved in the promoters of a subset of ciliary genes and a subsequent screen for this site in two Drosophila species identified novel RFX target genes that are involved in sensory ciliogenesis.

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.

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.

Development Molecular biology Genetics Genome studies

Background

Eukaryotic cilia and flagella are present 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 12345. Several human diseases are known to result from defects in ciliary assembly or function and have recently been designated as ciliopathies 5. Cilia are well-defined structures consisting of a microtubular axoneme composed of specific proteins that are assembled dynamically in a strict stereotypical pattern (for reviews, see 67). Ciliary assembly depends on intraflagellar transport (IFT) a dynamic process highly conserved in organisms ranging from the green algae Chlamydomonas to mammals (reviewed in 189). Several studies in various organisms have been instrumental in the identification of genes involved in the assembly and function of the cilium. The proteomic analysis of detergent-extracted ciliary axonemes from cultured human epithelial cells identified 214 proteins 10. More recently, a biochemical fractionation of Chlamydomonas reinhardtii flagella led to the identification of about 700 proteins, of which 360 had high confidence of truly being involved in flagellar composition 11. A proteomic analysis of Trypanosoma brucei flagella allowed the identification of 522 proteins 12. 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 et al. 13 and Li et al. 14 selected 187 and 688 genes, respectively, that are specific to ciliated organisms. Stolc et al. 15 used microarray hybridization to analyze induction levels of all C. reinhardtii 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 function of cilia and flagella.

Much less is known about the regulatory pathways that control the expression of ciliary components or direct 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 16. The transcription factor HNF1-β has also been shown to regulate several genes involved in ciliogenesis in the kidney 17. Most importantly, regulatory factor X (RFX) transcription factors play a key role in regulating genes involved in ciliogenesis. RFX transcription factors are conserved in a wide range of species, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and mammals. They share a characteristic DNA-binding domain of the winged-helix DNA binding family and bind to an X-box motif, an imperfect inverted repeat with variable spacing between the repeats 1819. Whereas only one Rfx gene is described in yeast and C. elegans, two Rfx genes are present in the Drosophila genome and five in mammals 20. Major clues on RFX functions in metazoans have been obtained from work on invertebrates. daf-19, the sole Rfx gene in C. elegans, is a key regulator of ciliogenesis 21. dRfx in Drosophila 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 2223. In mouse, we have shown that RFX function in ciliogenesis is conserved. Indeed, Rfx3 controls the growth of mouse embryonic node cilia 24 and Rfx3 loss-of-function leads to hydrocephalus with differentiation defects of ciliated ependymal cells of the choroid plexus and subcommisural organ 25. Moreover, Rfx3 mutant mice show insulin secretion failure and impaired glucose tolerance correlated with primary ciliary growth defects on islet cells 26. In zebrafish, Rfx2 is expressed specifically in multiciliated cells of the pronephros and loss of Rfx2 leads to cyst formation and loss of multicilia 27. The function of the other RFX proteins has yet to be linked to ciliogenesis. Rfx5, the most divergent mammalian member, regulates major histocompatibility class II gene expression and mutations in it are responsible for the bare lymphocyte syndrome 28. Rfx4 has been implicated in dorsal patterning of brain development in mice and may participate in circadian rhythm regulation in humans 29303132.

Because RFX function in ciliogenesis appears conserved from C. elegans 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 C. elegans have identified many genes involved in ciliogenesis 1421333435363738. 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 D. melanogaster and C. elegans and identify a first set of 14 RFX target genes. In particular, we show that all known Drosophila 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 control in D. melanogaster share conserved X-boxes with another divergent Drosophila species, D. pseudoobscura. Applied to the whole genome of both species, our comparative approach led to the identification of at least 11 novel RFX target genes. In vivo reporter assay studies for three of them confirmed their involvement in ciliary structure or function in Drosophila, thus illustrating the accuracy of our screen. In addition, we have established a highly confident Drosophila cilia and basal body (DCBB) gene list and highlight several genes as novel candidates for ciliogenesis. Our data are of particular importance for further genetic and genomic studies in the field of ciliogenesis and, consequently, for identifying genes involved in human ciliopathies.

Results

Homologs of C. elegans DAF-19 target genes are regulated by dRFX in Drosophila

Our previous work has shown that RFX transcription factors share a common function in ciliogenesis in worm and fly 2123. We thus inferred that an identical set of genes would be regulated by DAF-19 in C. elegans and dRFX in D. melanogaster. Indeed, among more than 20 previously identified DAF-19 targets expressed in all ciliated sensory neurons of C. elegans 21363738, we show that a majority of the homologous genes in fly are down regulated in dRfx mutants (Table 1). 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 have just differentiated. Moreover, the levels of expression of ciliary genes osm-6 and nompB, relative to the housekeeping gene TBP (TATA Binding Protein) or the pan-neural gene elav during pupae development, is at a maximum starting at 40 hours after puparium formation (data not shown). As shown in Table 1, 14 of 19 DAF-19 regulated genes for which a homologous gene can be found in Drosophila are also regulated by dRFX. Only one gene (CG5359/D1009.5/xbx-2/dylt-2) regulated by DAF-19 in all ciliated sensory neurons in C. elegans does not seem to be under dRFX regulation in Drosophila. Among all the C. elegans genes expressed and regulated by DAF-19 in a subset of ciliated sensory neurons, only CG9398/tulp appears to be under dRFX control in Drosophila. All the others, such as oseg3, NudC or amo, 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 time 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 CG13232/BBS4 in Drosophila, the only BBS gene that is not found in the C. elegans genome, and show that it is also down regulated 17-fold in a dRfx 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 retrograde dyneins, that carry particles that can be biochemically fractionated as A and B complexes 1. dRFX regulates genes encoding B complex components, but not A complex components.

Table 1

RFX target genes in C. elegans and D. melanogaster and in compartmentalized ciliogenesis

D. melanogaster gene ID (name)

Homologs in vertebrates or Chlamydomonas

Fold variation

Ciliary type [13]

C. elegans gene ID (name)

DAF-19 control in C. elegans

Downregulated >2 fold

CG1126

BBS5

95.2*

R01H10.6 (bbs-5)

All [14]

CG3769

D2LIC/LIC3

63*

F02D8.3 (xbx-1)

All [21,35]

CG4525

Novel

223.6*

C27H5.7 (dyf-13)

All [37]

CG8853

IFT55/hippi

21.8*

F59C6.7 (che-13)

All [33]

CG9333 (oseg5)

WDR56

4.4*

F38G1.1 (che-2)

All [21]

CG9595 (osm-6)

NDG5

22.2*

R31.3 (osm-6)

All [21]

CG12548 (nompB)

TG737

12.7 [92]*

Y41g9a.1 (osm-5)

All [34]

CG13691 (BBS8)

BBS8

2.7*

T25F10.5 (bbs-8)

All [36]

CG13809 (oseg2)

IFT172/wim

9.7*in vivo

T27B1.1 (osm-1)

All [21]

CG14825 (BBS1)

BBS1

211*

Y105E8A.5 (bbs-1)

All [36,37]

CG15666

BBS9

20*

C48B6.8

All [37]

CG17599

QILIN

29.5*

C04C3.5 (dyf-3)

All [93]

CG9398 (Tulp)

Tubby

3.2^‡

F10B5.4 (tub-1)

Subset [36]

CG3259

Traf3ip1/MIP-T3

In vivo

C02H7.1

CG5142

Novel

16.4*

F54C1.5a (dyf-1)

CG7735

BBS3

40.9*

C38D4.8 (arl-6)

CG9227 (tectonic)

tectonic

In vivo

Y38F2AL.2

CG14367

Novel

10.2^†

Y108G3AL.3

CG14870

B9 domain

3.1*

K03E6.4

CG15161

IFT46

207.1*

F46F6.4 (dyf-6)

CG18631

Novel

14.2*

K07G5.3

CG13232 (BBS4)

BBS4

17*

Downregulated <2-fold

CG30441

IFT20

2^‡

Y110A7A.20

All [37]

CG1399-PB

LRRC16

1.8^†

K07G5.1

CG11048

Rib74

1.5^†

Y49A10A.1

CG13178

Novel

1.7^†

R10F2.5

Overexpressed

CG2006

Dtwd1

0.5*

Y53C12A.3

Invariant in Drosophila

CG5359

Tctex

D1009.5 (xbx-2/dylt-2)

All [36]

CG11237 (oseg6)

wdr19

ZK520.3 (dyf-2)

Subset [94]

CG6504 (amo)

PKD2

0.9

Y73F8A.1 (pkd-2)

Subset [95]

CG9710 (nudC)

NudC

0.7

F53A2.4 (nud-1)

Subset [36]

CG11838 (oseg3)

IFT140

1.4

C27A7.4 (che-11)

Subset [36]

CG2069 (oseg4)

WDR35

C54G7.4 (ifta-1)

CG6560

arl3

F19H8.3 (arl-3)

CG7161 (oseg1)

IFT122

1.4 in vivo

F23B2.4 (daf-10/osm-4)

CG10642 (Klp64D)

KIF3A

1.1

Y50D7A.6 (klp-20)

CG11755

Novel

1.1

D2089.3

CG11759 (Kap3)

KIFAP3

F08F8.3 (kap-1)

CG5195

L domain like

1.2

CG31249

Novel

0.9

CG32392

rshl2

1.1

Not determined in Drosophila

CG3798 (Nmda1)

F40F9.1 (xbx-6)

Subset [36]

CG9310 (Hnf4)

HNF4A/G

T19A5.4 (nhr-44)

Subset [36]

CG17228 (pros)

PROX1/2

K12H4.1 (ceh-26)

Subset [95]

Not conserved in Drosophila

Y75B8A.12 (osm-12/bbs-7)

All [36]

F20D12.3 (bbs-2)

All [36]

M28.7 (nph-1)

All [96]

R13H4.1 (nph-4)

All [96]

C47E8.6

All [37]

ZK328.7a

All [37]

Y102E9.1 (odr-4)

Subset [36]

C23H5.3 (xbx-4)

Subset [36]

M04D8.6 (xbx-3)

Subset [36]

T24A11.2 (xbx-5)

Subset [36]

R148.1 (xbx-7)

Subset [36]

D2005.2 (nlp-8)

Subset [95]

*p value < 0.001; ^†0.001 <p value < 0.05; ^‡0.05 <p value < 0.08. Dashes indicate not applicable. Cp, compartmentalized ciliogenesis; ND, not determined.

Genes specific to compartmentalized ciliogenesis are regulated by dRFX in Drosophila

Interestingly, most of the genes regulated by dRFX also fall in the list of genes for compartmentalized ciliogenesis (Cp ciliary type, Table 1) defined by the work of Avidor-Reiss et al. (Table 1) 13. This group of genes is found only in genomes of species showing compartmentalized cilia biogenesis, but neither in the genomes of non-ciliated organisms nor in Plasmodium falciparum, which uses cytosolic cilia biogenesis. We thus tested the expression of almost all the genes described in the Cp category in control and dRfx deficient Drosophila. Among the 34 Cp ciliary genes tested by real-time PCR, 18 were down regulated more than 2-fold in a dRfx mutant background, 4 were significantly reduced between 1.5- and 2-fold and one was significantly over expressed. Eleven genes did not show significant expression variations between control and mutant background (Table 1).

In order to demonstrate the accuracy of our quantification procedure, we performed in vivo observations of reporter constructs of some of the genes in wild-type and dRfx deficient backgrounds (Figure 1). As previously published, sensory neuron ciliary endings are missing in a dRfx deficient background 23. As observed in the cell body or remaining dendrite, the expression of osm-1 is totally shut down in the dRfx deficient background, whereas the expression of oseg1 is not affected (Figure 1), in agreement with real-time RT-PCR results. Interestingly, CG3259 and CG9227 cDNAs were hardly detectable by real-time PCR and, thus, difficult to quantify. However, in vivo observations of reporter constructs in wild-type and dRfx mutant backgrounds show a complete absence of expression of these two genes in the mutant background (Figure 1).

Figure 1

In vivo observations of reporter constructs in control or dRfx-deficient Drosophila

In vivo observations of reporter constructs in control or dRfx-deficient Drosophila. (a) Schematic of two typical chordotonal organs of the Drosophila leg or antenna. The different segments of the dendrite and of the ciliated ending are shown. Sensory neurons have a single cilium (arrow) extending from their dendrite (arrowhead). (b) Live confocal image of GFP driven expression of osm-1 transgene in a control femur. (c) GFP expression is totally shut down in a dRfx mutant background. (d-i) Confocal imaging of chordotonal neurons labeled with anti-ELAV (red) and anti-GFP (green). oseg1-GFP expression in (d) control flies and (e) a dRfx mutant background. Note that oseg1-GFP expression is not affected in the mutant background. CG3259-GFP expression in (f) control flies and (g) dRfx mutant flies. Reporter construct expression is totally shut down in the mutant background. Johnston's organs from antennae of adult flies carrying CG9227-GFP transgenes in (h) control and (i) dRfx mutant pupae. Note the absence of expression in the mutant background. Scale bar = 10 μm.

In summary, we show that RFX target genes are mainly conserved between C. elegans and D. melanogaster. Our functional comparative approach between both organisms combined with the work of Avidor-Reiss et al. in Drosophila allowed us to identify 27 genes that are regulated by dRFX in Drosophila. A majority of them are shown to be involved in ciliogenesis.

X-box conservation between D. melanogaster and D. pseudoobscura

As previously described 13142136373839, the X-box promoter motif has been used successfully to screen for genes involved in ciliogenesis. As shown above, this first set of X-box gene data in Drosophila is thus a key to better understand the link between X-box sequences and dRFX transcriptional control in Drosophila. We looked for X-boxes in the promoters of dRFX target genes. We searched for X-boxes up to 3 kb upstream of the ATG for each of them, with the most degenerated X-box consensus deduced to date from known RFX protein binding sites (RYYNYY N1-3 RRNRAC). We could identify several X-boxes for each gene (Table 2, columns 2 and 3). However, known negative control genes also presented X-boxes at the same frequency and no particular constraint on the consensus seemed to correlate with one set of genes. Therefore, the presence for one gene of an X-box upstream of its ATG is not predictive of dRFX-dependent expression. We thus turned to the D. pseudoobscura genome. The two Drosophila species' most recent common ancestor occurred 40-60 million years ago. The average identity of coding sequence between D. melanogaster and D. pseudoobscura at the nucleotide level is 70% for the first and second bases of codons, and 49% for the wobble base. Intron sequences are 40% identical, untranslated regions 45-50%, and DNA protein binding sites extracted from the literature have been estimated to an average of 63% 40. Moreover, detailed comparison of both Drosophila genomes showed that 50-70% of known DNA binding sites reside in conserved sequence blocks in the genomes, called conserved regulatory elements (CREs), whereas the overall conservation of the cis-regulatory regions is low 414243.

Table 2

X-box comparisons in promoters of dRFX regulated genes, between Drosophila melanogaster and Drosophila pseudoobscura

D. melanogaster

D. pseudoobscura

D. melanogaster

D. pseudoobscura

No. of X-box

Strand

Conserved blocks around X-box^†

Gene ID

No. of conserved X-box*

X-box

Dist.

X-box

Dist.

100 bp

25 bp

Genes down regulated in dRfx mutant

CG1126-PA

GA10872-PA

1^‡

GTTGCC T AGCAAC

CG3259-PA

GA17011-PA

1^‡

GTTGCC AG GACAAC

GTTGTC AG GACAAC

CG3769-PA

GA17674-PA

1^‡

GTTGCT AGT AGCAAC

GTTGCC AG GACAAC

CG4525-PA

GA18233-PA

1^‡

GTTGCC A AGCGAC

134

GTTGCC A AGCGAC

239

CG8853-PA

GA21369-PA

1^‡

GTTACC TT GGCGAC

GTTACC AT GGAAAC

110

CG9227-PA

GA21627-PA

1^‡

GTTACT TT GACAAC

119

GTTGCC AG AGCAAC

146

CG9595-PA

GA21901-PA

1^‡+ 2

GTTGCC G GGCAAC

126

GTTGTC CG GGCAAC

141

ATTTTT GTT AGCAAC

264

ACTTTT GC AAAAAC

699

GCTGTT ACA AGAGAC

2,969

GCTGCT GCA GGAAAC

2,671

CG12548-PA

GA11690-PA

ATCACC AA GGCAAC

2,335

ATCACC TT GGAAAC

868

GCCTTT C GGAGAC

2,833

GCCGCT T GATGAC

2,638

CG13178-PA

GA12098-PA

GCCGTT AGC AAGAAC

2,551

GCCACC AGG AAAAAC

2,106

CG13809-PA

GA12544-PA

1^‡

GTTGCC AC AACAAC

123

GTTGCC AC AACAAC

103

CG15161-PA

GA13541-PA

2^‡

GTTGTC AG GACGAC

321

GTTGTC AA GACAAC

311

GTTGTC AG GACGAC

321

GTTTTT GCA GGCAAC

391

CG30441-PA

GA15848-PA

1^‡

GTTGTC AAT AGCAAC

GTTGTC TGT GACAAC

122

CG17599-PA

GA14573-PA

1^‡

GTTACC T AGCAAC

141

GTTGCC T GGCAAC

206

CG18631-PA

GA15024-PA

1^‡

GTTGCC CAT GGCAAC

2,731

GTTGCC GTT AGCAAC

2,633

CG15666-PA

GA13881-PA

1^‡

GTTGCC AA GGCAAC

GTGGCC AT GGCAAC^§

CG14870-PA

GA13310-PA

1^‡

GTCTCC CG GGCAAC

-22

GTATCC TG GGCAAC^§

-6

CG1399-PB

GA12678-PA

CG5142-PA

GA18687-PA

CG9333-PA

GA21709-PA

CG9398-PA

GA21760-PA

CG11048-PA

GA10726-PA

CG13691-PA

GA12462-PA

CG14367-PA

GA12937-PA

Invariantly expressed genes in dRfx mutant

CG5195-PA

GA18727-PA

CG5359-PA

GA17011-PA

1^‡

GTTATT CGT GGTGAC

141

GTTGTT G AGGAAC

CG6504-PA

GA19567-PA

CG6560-PA

GA19685-PA

CG7161-PA

GA20145-PA

CG9710-PA

GA21982-PA

CG10642-PA

GA10463-PA

1^‡

GTCGTT TAA GGAAAC

1,041

GTTGTC ATG AGTAAC

1,226

CG11755-PA

GA11176-PA

ACCGCC CAG AAGAAC

2,418

ACTACC GT GAAAAC

1,576