1 Karolinska Institute, Department of Biosciences and Södertörn
University College, Section of Natural Sciences, S-14189 Huddinge,
Sweden
2 Department of Genome Sciences, University of Washington, Seattle, WA 98195,
USA
3 Department of Microbiology, University of Washington, Seattle, WA 98195,
USA
4 Department of Molecular Biology, Massachusetts General Hospital and Department
of Genetics, Harvard Medical School, Boston, MA 02114, USA
5 Department of Molecular Biology and Biochemistry, Simon Fraser University,
Burnaby, British Columbia V5A 1S6, Canada
* Author for correspondence (e-mail: peter.swoboda{at}biosci.ki.se)
Accepted 10 February 2005
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SUMMARY |
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By using a genome search approach for X-box promoter motif-containing genes (xbx genes) we identified a list of about 750 xbx genes (candidates). This list comprises some already known ciliary genes as well as new genes, many of which we hypothesize to be important for cilium structure and function. We derived a C. elegans X-box consensus sequence by in vivo expression analysis. We found that xbx gene expression patterns were dependent on particular X-box nucleotide compositions and the distance from the respective gene start. We propose a model where DAF-19, the RFX-type transcription factor binding to the X-box, is responsible for the development of a ciliary module in C. elegans, which includes genes for cilium structure, transport machinery, receptors and other factors.
Key words: X-box, DAF-19, Ciliary genes, C. elegans
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Introduction |
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In the nematode C. elegans 60 of the 302 neurons of the
hermaphrodite are ciliated sensory neurons (CSN)
(Ward et al., 1975;
White et al., 1986
), forming
many structurally distinct types of sensory cilia. Whereas many ciliary
mutants are available in C. elegans, there is only one known gene
mutation that completely eliminates all classes of sensory cilia and all
functional components of cilium structure. This gene is daf-19, and
it encodes the sole C. elegans member of the RFX-type transcription
factors (Swoboda et al.,
2000
), found widely in the eukaryotic kingdom. All members of the
RFX transcription factor family are characterized by the presence of a
conserved DNA binding domain (DBD). The RFX-DBD binds to special motifs
(X-boxes) in promoters of its target genes. Genes containing the X-box
promoter motif are called xbx genes.
Apart from C. elegans DAF-19, the RFX family currently contains
nine characterized members: five in mice and humans (RFX1-5)
(Emery et al., 1996a), two in
Drosophila melanogaster (dRFX1-2)
(Dubruille et al., 2002
;
Otsuki et al., 2004
), and one
member each from Schizosaccharomyces pombe
(Wu and McLeod, 1995
) and
Saccharomyces cerevisiae (Huang
et al., 1998
). The data obtained so far suggest diverse biological
roles of RFX proteins. In yeasts they regulate some aspects of the cell cycle
(Wu and McLeod, 1995
;
Huang et al., 1998
). In humans
RFX factors are involved in the transcriptional regulation of major
histocompatibility complex class II genes (RFX5)
(Reith and Mach, 2001
) and in
the modulation of Ras signaling in epithelial cells (RFX3)
(Maijgren et al., 2004
).
The finding of X-boxes in promoters of ciliary genes in C. elegans
has revealed an important role of the RFX family in the regulation of
ciliogenesis (Swoboda et al.,
2000). Since then, the conservation of RFX-binding elements has
been reported in several distantly related species. For example, some ciliary
genes in D. melanogaster contain X-box-like sequences in their
promoters (Avidor-Reiss et al.,
2004
). However, an experimental demonstration of an RFX-dependence
for Drosophila ciliary gene candidates exists so far only for the
nompB gene (Han et al.,
2003
). Recently, data about the possible RFX regulation of
ciliogenesis in mammals were also obtained. Rfx3-deficient mice
exhibit frequent left-right asymmetry defects, which are caused by ciliary
abnormalities in mutant embryos (Bonnafe et
al., 2004
). Mouse RFX3 regulates the expression of D2lic,
the mouse ortholog of the C. elegans ciliary gene xbx-1, but
does not affect the expression of Tg737, the mouse ortholog of the
C. elegans ciliary gene osm-5. These observations suggest
that RFX regulation of ciliogenesis in higher organisms is more complicated,
and different subtypes of RFX proteins may be restricted to particular
components of ciliary structure.
In our current work we first concentrated on the isolation of genes
important for cilium structure and function using a genome-wide X-box promoter
motif search in the nematode species C. elegans and C.
briggsae. In this computational approach we focused our efforts only on
5' flanking regions of genes, since X-box motifs have previously been
shown to be functional in those regions
(Swoboda et al., 2000).
Subsequently, we performed expression analyses of the group of positive C.
elegans matches in wild-type and daf-19 mutant backgrounds,
together with X-box mutagenesis experiments. Results of these analyses
established the X-box consensus for C. elegans, the approximate
number of xbx genes in the C. elegans genome and assigned
already known and newly found ciliary genes to specific structural and
functional groups. Because the organization of C. elegans sensory
cilia is very similar to sensory cilia in mammals, the results obtained with
the C. elegans model will have general significance.
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Materials and methods |
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Promoter motif search algorithm and sequence analyses
The X-box motif search was performed primarily with a Perl-based algorithm
that searches through a given genome sequence for all possible matches. The
algorithm first finds all sequences that match a defined consensus. After that
step, the main module of the program implements a cross-match file (P. Green,
personal communication), which compares a 3 kb window downstream of each match
to a file containing the DNA sequences for all predicted genes, and a file
containing assembled ESTs. Cross-match parameters `minmatch' and
`minscore' were set to 40. All other parameters were kept at default values.
Minimal and maximal distances from positive matches to predicted genes were
set to a range of 0-1000 nucleotides. To obtain a copy of the algorithm,
contact
kbubb{at}u.washington.edu.
Cross-match must be obtained separately (see
www.phrap.com
for access/download information).
Subsequent to genome analyses using Cross-match, we made use of another program, DNA Motif Searcher, which takes a set of user-definable X-box sequences to search for additional motif instances in the genome. The set of motifs is interpreted into a position-specific score matrix (PSSM). Using this PSSM, the program can then identify the closest matching occurrences of the motif based on a score cutoff, or it can identify the top number of occurrences of the motif. For download or for more detailed explanations about Motif Searcher, please contact jht{at}u.washington.edu.
Genome sequence information, EST files, gene predictions and identities for X-box searches were obtained from the following sources: C. elegans complete genome sequence, WS122 release (ftp://ftp.sanger.ac.uk/pub/wormbase/WS122); C. briggsae draft genome sequence, cb25.agp8 version (ftp://ftp.wormbase.org/pub/wormbase/briggsae).
Generation and analysis of expression constructs
GFP expression constructs were designed by inserting about 2 kb of promoter
regions and the first several codons of a gene of interest into the GFP
expression vector pPD95.77 (gift from A. Fire). PCR fragments of promoter
regions were obtained from wild-type N2 genomic DNA and cloned into
appropriate sites of pPD95.77. For some genes, the wild-type X-boxes within
promoters were mutated by overlap extension mutagenesis, replacing X-box
sequences with nonspecific nucleotides containing indicative restriction
enzyme sites. To check for correct translational reading frames and promoter
regions, junctions between vector and amplified inserts were verified by
sequencing for all constructs. For the XBX-2::GFP translational fusion, the
entire coding sequence of the gene with about 1 kb of promoter were fused to
the pPD95.77 vector.
The following worm strains were used for injections and expression analyses: JT8651, JT6924 and JT204. The strain JT8651 daf-19(m86)/mnC1; lin-15(n765ts) served as the wild-type background, since daf-19(m86) is fully recessive. daf-19 mutants are strongly Daf-c (dauer larva formation constitutive) across the normal temperature range. Therefore, segregating dauers were recovered at 15°C to obtain a daf-19 homozygous background. Alternatively, the strain JT6924 daf-19(m86); daf-12(sa204) was used as a daf-19 mutant background. Worms of this genotype exhibit a Daf-d (dauer larva formation defective) phenotype and do not require the recovery of dauers. In this case, JT204 daf-12(sa204) worms were used as a wild-type background with regard to daf-19.
Adult hermaphrodites were transformed using standard protocols
(Mello et al., 1991).
Constructs were injected typically at 10-100 ng/µl along with coinjection
markers such as pRF4 (contains the dominant marker rol-6(su1006)) or
pBLH98 (contains the wild-type lin-15 gene to rescue
lin-15(n765ts)).
Microscopy and imaging
GFP expression patterns were analyzed in stable transgenic lines at
1000x magnification by conventional fluorescence microscopy (Zeiss
Axioplan 2). Expression patterns were examined in at least two independent
transgenic lines at most developmental stages of the worm. Neuronal cell
anatomies and identities followed published descriptions
(Ward et al., 1975;
White et al., 1986
).
For the analysis of XBX-2::GFP movement properties, worms were mounted on agarose pads and anesthetized with 10 mM levamisol. Adult worms were analyzed with a Leica confocal imaging spectrophotometer TCS SP unit mounted on a Leica DMIRBE inverted microscope, and the obtained images were processed using Leica Confocal Software 2.5. Images were taken with a 63x objective and a 488 nm GFP filter. At least 40 stacked images were converted into an AVI file with a rate of two frames per second.
RNAi feeding experiments and fluorescent dye filling assays
RNA-mediated interference (RNAi) was performed according to standard
methods (Timmons et al.,
2001). PCR fragments for genes of interest were generated from N2
genomic DNA and cloned into the double T7 promoter-containing vector L4440
(gift from A. Fire). All constructs were transformed into HT115(DE3) bacterial
cells and plated onto NGM plates with antibiotics and IPTG. L4-stage
hermaphrodites were transferred to plates with induced bacteria and
F3 progenies were analyzed for possible phenotypes.
Fluorescent dye-filling assays were performed essentially as described
previously (Starich et al.,
1995) using the fluorescent dye DiI. Worm strains N2 and CB3323
were used as positive and negative controls, respectively. Stained adult
hermaphrodites were analyzed at 1000x magnification by conventional
fluorescence microscopy (Zeiss Axioplan 2).
Genetic characterization of xbx gene mutants
All deletion alleles analyzed in this study were generated by the C.
elegans Gene Knockout Consortium
(http://celeganskoconsortium.omrf.org/)
using publicly available methodology
(http://www.mutantfactory.ouhsc.edu/protocols.asp).
The original mutated strains RB819 xbx-4(ok635) IV and RB957 xbx-6(ok852) V were outcrossed three times with N2 and the following worm strains: JT7146 egl-4(n478) unc-33(e204) IV and DR108 dpy-11(e224) unc-42(e270) V, respectively. Outcrossed worms resulted in homozygous mutant strains containing the xbx-4(ok635) IV and xbx-6(ok852) V deletion alleles. These strains were then used as the basis for further analysis.
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Results |
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Expression patterns of orthologous genes are often conserved. Because many
orthologous transcription factors are also functionally conserved, one
possible model to account for homologous gene expression patterns is
conservation of specific binding sites within regulatory elements of
orthologous genes (Ruvinsky et al., 2003). The nematodes C. briggsae
and C. elegans are closely related species with very similar overall
genome organizations (Stein et al.,
2003). To find possible conservations of X-box regulatory elements
between those two organisms we applied the C. elegans X-box search
strategy to the C. briggsae genome. The number of obtained matches
was slightly less than with C. elegans, probably because of the draft
quality of the C. briggsae genome. Nevertheless, the profile of X-box
distribution within promoter regions was similar to that of C.
elegans (Fig. 1D).
In summary, using two different X-box promoter motif search approaches, X-box consensus sequences with varying degrees of refinement, together with cross-species comparisons and gene expression analysis (see below), we were able to identify a large number of bona fide xbx genes, a significant part of which we expect to also be ciliary- or CSN-specific genes.
Expression analysis of the xbx gene candidates
Our computational search has revealed a large, heterogeneous group of X-box
matches (see Table S1 in supplementary material). To determine the specificity
of our computational search with regard to ciliary and CSN structure and
function we initiated expression analyses for some of the genes. For this
purpose we isolated a group of candidates with different nucleotide
compositions of the X-box motifs, different positions within promoter regions
and different proposed molecular functions of matching genes.
Our previous model associated the expression of xbx genes with CSN
(Swoboda et al., 2000). We
predicted that DAF-19 function, a particular X-box composition and its
position within the promoter should be required for the expression of this
group of genes. Therefore, expression patterns of selected genes were analyzed
in both wild-type and daf-19 mutant backgrounds. For some genes we
also analyzed the actual X-box sequence by replacing it with nonspecific
nucleotides within the respective expression construct.
Based on our obtained expression data (Table 1), we subdivide xbx genes into groups: (1) genes that are strongly regulated by DAF-19 and required for the general development of cilia; (2) genes that are partially regulated by DAF-19 and are probably required for more specific ciliary and/or CSN functions.
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Most genes from this group encode known participants of intraflagellar
transport (IFT): osm-1, osm-5, osm-6, che-2, che-13, xbx-1. The
mechanism of IFT was originally described in the biflagellate alga
Chlamydomonas reinhardtii
(Kozminski et al., 1993). It
is characterized by the movement of IFT particles along ciliary/flagellar
axonemal microtubules by means of kinesin and dynein motor molecules. C.
elegans xbx genes encoding IFT proteins are well described. IFT
components such as OSM-1, OSM-5, OSM-6, and CHE-13 are associated with the
heterotrimeric motor protein kinesin-II. They are essential for anterograde
transport (Signor et al.,
1999
; Haycraft et al.,
2001
; Haycraft et al.,
2003
). The gene xbx-1 encodes a dynein light intermediate
chain (DLIC) that is important for retrograde transport within cilia
(Schafer et al., 2003
).
Herein we report a new IFT gene, xbx-2 (D1009.5). The
xbx-2::gfp promoter fusion was strongly expressed in most of the
ciliated sensory organs of the worm amphids, phasmids, inner and outer
labial quadrants (Fig. 3A). The
XBX-2 protein contains a Tctex-1 domain that belongs to the family of dynein
light chain (DLC) proteins. These molecules are essential for dynein assembly
and participate in specific motor-cargo interactions
(DiBella et al., 2001;
Tai et al., 2001
). To analyze
the possible role of XBX-2 in the IFT process, we generated transgenic worms
expressing XBX-2::GFP protein. Using time-lapse confocal microscopy, we
observed movement of XBX-2::GFP particles along the ciliary axoneme in both
retrograde (Fig. 4) and
anterograde directions (see also Movie 1 in supplementary material).
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The second group of xbx genes includes many novel genes, which are
expressed in various subsets of CSN. For example, xbx-3::gfp was
strongly expressed in all amphid and phasmid neurons, but not in other
ciliated sensilla (Fig. 3B). In
daf-19 mutant worms, expression was restricted to one amphid neuron
and abolished in phasmids, with occasional ectopic expression in other
tissues. Expression of the novel gene xbx-4 was also observed in some
amphid and phasmid neurons (Fig.
3C), but in daf-19 mutants it was completely abolished in
both organs. The xbx-5::gfp promoter fusion was characterized by
faint, punctate expression in phasmids and some amphid neurons
(Fig. 3D). In a daf-19
mutant background expression was absent in amphids but still visible in
phasmids. The predicted XBX-5 protein contains seven transmembrane domains and
can be considered as a possible receptor. The xbx-6::gfp construct
was abundantly expressed in many cell types: pharyngeal muscles, numerous
neurons in the head and tail regions, the ventral nerve cord and body wall
muscles. Because of high overall expression levels, we were not able to
identify individual CSN in the head region, but we observed expression in
phasmids (Fig. 3E), which was
strictly DAF-19 dependent. The expression in other cells was unchanged in
daf-19 mutants. The xbx-6 gene encodes an
N-methyl-D-aspartate receptor-associated protein. The novel gene
xbx-7 was expressed in phasmids, some amphid neurons and in
interlabial neurons (Fig. 3F),
while in daf-19 mutants expression was significantly reduced. The
gene che-11 encodes a large protein, which is orthologous to
Chlamydomonas IFT140 (Qin et al.,
2001). The expression level of che-11::gfp was
significantly reduced in phasmids and amphids in daf-19 mutants
(Table 2), while in the labial
neurons it was almost unchanged. The nuclear hormone receptor gene
nhr-44 was expressed in head neurons, including the ciliated sensory
neuron ASK, and also in other cell types, and its overall expression
properties fit those of other xbx genes
(Table 1).
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The gene odr-4 has been shown to be important for the localization
of some seven transmembrane domain odorant receptors to cilia
(Dwyer et al., 1998). The
expression of an ODR-4::GFP translational fusion was significantly reduced
both in daf-19 mutants and after mutation of the X-box sequence (data
not shown). This indicates that not only transport mechanisms within cilia
(IFT), but also to cilia (ODR-4) are under DAF-19 control.
The C. elegans ortholog of the NudC gene of the fungus
Aspergillus nidulans, nud-1, was identified as a candidate
xbx gene during our search. NUD-1 is an important component in
microtubule-dependent nuclear positioning, which is required for proper
growth, development and cellular function in both lower and higher eukaryotes.
Sustained expression of nud-1::gfp in CSN was previously described
(Dawe et al., 2001). We
introduced this construct into a daf-19 mutant background and found
its expression drastically reduced (Table
2).
The gene tub-1 is the worm ortholog of murine tubby
which, when mutated, leads to neuronal deficits and late-onset obesity
(Carroll et al., 2004).
tub-1 mutant worms exhibit functional defects in CSN and show a mild
elevation of lipid accumulation (H.Y.M., unpublished data). The translational
GFP fusion for tub-1 was expressed only in the cytoplasm in all CSN
(Fig. 1G). The expression level
of this construct was significantly reduced in a daf-19 mutant
background (Table 2). We
analyzed the 5'-UTR region of the human TUB gene and found two
X-box-like sequences that perfectly match the C. elegans consensus:
GTTGCC AT GGAAAC (296) and GTTGCT AT AGTAAC (339). Intriguingly,
microtubule-associated protein 1A (MAP1A) can modify hearing defects in
tubby mice (Ikeda et al.,
2002
), and its expression is regulated by RFX molecules
(Nakayama et al., 2003
). These
observations suggest that the regulation of tubby pathways by RFX
transcription factors can be conserved in evolution. Based on the data from
the second group of xbx genes, we conclude that DAF-19 only partially
regulates the expression of certain genes. These genes may only be required
for specialized functions in CSN or during ciliogenesis.
The remaining genes analyzed were mostly expressed in many other different cell types and only in very few cases was expression observed predominantly in CSN (Table 1; see Table S2 in supplementary material). We checked some genes from this group (zag-1, aqp-2, gpa-9, F55D12.1, F17A2.3) in a daf-19 mutant background and found that expression patterns were unchanged in the absence of DAF-19 function. Most of these X-box matches differ from the refined consensus or are located further upstream of the ATG (Table 1).
An additional confirmation and filtering mechanism used was a cross-species comparison, where candidates were classified as likely or unlikely xbx genes depending on the conservation of the X-box motif sequence between C. elegans and C. briggsae (Table 1). We found that most of the genes from the first group have putative C. briggsae orthologs with nearly identical X-box sequences and positions in their promoters. In the second group, the frequency of X-box occurrences in C. briggsae orthologs was reduced. C. elegans xbx gene candidates that were expressed predominantly in cell types other than CSN typically have no X-box-like sequences in promoters of their C. briggsae orthologs (Table 1; see Table S2 in supplementary material). Thus, the actual X-box sequence and position conservation between C. elegans and C. briggsae (and maybe other organisms) can be used as an additional measure to deduce possible molecular roles of xbx genes in cilia or CSN.
The expression data from 22 GFP fusions that show dependence on DAF-19 function and actual X-box sequence (Table 1) allowed us to derive an in vivo refined consensus for C. elegans xbx genes (Fig. 5A). This consensus can be characterized as a 14 nt imperfect palindromic sequence (GTHNYY AT RRNAAC) consisting of conserved nucleotides at 5' (GT) and 3' (AC) ends of the two half-sites separated by a conserved AT spacer. Most of the X-boxes matching this consensus are located in immediate proximity to the gene start (in the range of 50-250 bp upstream of the ATG). Lack of DAF-19 function or changes in the actual X-box sequence lead to drastic reduction or variation of gene expression patterns, suggesting a crucial role of the given motif sequence in the regulation of target genes (see also Discussion).
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xbx genes from group 1 show Dyf and to various extents also
different sensory phenotypes (Table
1) (Collet et al.,
1998; Signor et al.,
1999
; Fujiwara et al.,
1999
; Haycraft et al.,
2001
; Haycraft et al.,
2003
; Schafer et al.,
2003
; Blacque et al.,
2004
; Li et al.,
2004
). Mutations that reduce dye filling of amphid and phasmid
neurons are indicative of general defects in cilium structure and are often
accompanied by various sensory mutant phenotypes
(Starich et al., 1995
).
The availability of a large group of xbx gene candidates obtained
in our computational search prompted us to try a screening approach for new
members of general cilium structure and function using genetic interference
mediated by double stranded RNA (RNAi). We analyzed already known dyf
genes (che-13, osm-5) together with novel candidates (bbs-2,
bbs-7, xbx-1, xbx-2) with regard to the Dyf phenotype. It was known that
RNAi is less efficient in neuronal types of cells
(Simmer et al., 2002). To
increase possible effects of interference, in parallel to wild-type worms we
also tested rrf-3 mutants, which are sensitive to RNAi in diverse
tissues, especially in neurons (Simmer et
al., 2002
). We found that RNAi of the ciliary genes tested did not
result in strong Dyf phenotypes in wild type or rrf-3 mutants (data
not shown). Therefore, RNAi cannot be implemented as a quick and easy
screening technique with regard to the Dyf phenotype in C.
elegans.
Unlike those in group 1, the roles of many genes in group 2 are largely unknown. To begin the functional investigation of the second group we analyzed two genes, xbx-4 and xbx-6, mutants of which were available. The xbx-4(ok635) deletion extends over 951 bp starting in the promoter region and ending in the first intron, completely eliminating the beginning of the gene. The xbx-6(ok852) deletion extends over 1720 bp starting in the promoter region and covering five of the six exons of the gene. Since the expression of these genes is associated with CSN, we first focused our efforts on phenotypes related to defects in cilium structure or sensory abnormalities. The following results were obtained: both analyzed deletion alleles demonstrated wild-type responses with regard to fluorescent dye filling, high osmotic strength avoidance and in odorant sensation assays using three different odors (data not shown).
These xbx-4 and xbx-6 results suggest that in some
instances members of the xbx gene family may have specialized
molecular functions and therefore mutants have more specialized sets of
sensory phenotypes, although we cannot formally exclude genetic redundancy
with other (xbx) genes expressed in CSN. For example, two other
members of group 2, the genes odr-4 and tub-1, when mutated,
also do not produce general structural defects of cilia, but more specialized
functional ciliary abnormalities, such as selective defects in odorant
sensation (Dwyer et al., 1998)
(H.Y.M., unpublished).
In conclusion, our data support the sorting of xbx genes into different groups, where members of group 1 are typically required for more general aspects of cilia formation, while genes from group 2 are typically required for more specialized functions within cilia and/or CSN.
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Discussion |
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We note that our search is not completely unbiased, because the search algorithm is based on an originally small set of experimentally proven xbx genes. Therefore, as further xbx genes will be shown to be ciliary- or CSN-specific genes, their X-boxes will be included into the search parameters (bootstrap mechanism).
Other genome search approaches, in part utilizing X-box matches as a
parameter, were used in different organisms to find general components of
cilia formation (Li et al.,
2004; Avidor-Reiss et al.,
2004
), yielding a set of conserved ciliary genes and gene
candidates. We compared information from flagellar and basal body genes
(Li et al., 2004
) with our
list of X-boxes and found an overlap of 15 X-box matches. At the same time, we
found eight additional xbx gene candidates (Table S1 in supplementary
material). Despite overlaps, many of the X-box matches were different between
the respective searches, possibly because of different experimental
parameters. Another important comparison was obtained from a recent study of
olfactory neuron-specific genes (Colosimo
et al., 2004
), where we found 56 genes in common (Table S1 in
supplementary material). All approaches together with further filtering
mechanisms will give a complete, exhaustive list of genes important for
structure and function of cilia and CSN.
Properties of the X-box motif in C. elegans
cis-Regulatory elements are information processing devices
hardwired into the genomic DNA sequence, the function of which is to regulate
gene expression (Davidson,
2001). Frequently, they are organized into modules that include
many sites for DNA binding proteins
(Howard and Davidson, 2004
).
In C. elegans, only some cell-type-defining transcription factors
(CEH-10/TTX-3, MEC-3) target single binding sites in promoters of regulated
genes (Zhang et al., 2002
;
Wenick and Hobert, 2004
).
Typically, also DAF-19 targets contain only a single X-box motif in their
promoters. Nevertheless, different xbx genes show different
expression properties, suggesting the presence of specific DAF-19
co-regulators. We propose that the information about particular gene
expression profiles could already be stored at the level of the X-box
sequence. For example, all genes for cilium structure and IFT from group 1
have perfect matches to the refined X-box motif consensus. Each gene of this
group is expressed in most or all CSN and is strongly dependent on DAF-19
function. Whereas a perfect match to the `refined consensus' does not
automatically predict expression in all CSN, variations in the X-box motif
sequence, especially in the more variable half-site (GTHNYY), predict
different gene expression properties (xbx-3, che-11, odr-4, tub-1).
These genes are either expressed only in a subset of CSN (xbx-3,
odr-4) or are only partially dependent on DAF-19 function (che-11,
tub-1).
Structural experiments have shown that each half-site of a symmetric X-box
interacts with both DBDs of the RFX homodimer
(Gajiwala et al., 2000).
Nevertheless, binding of RFX is not ultimately dependent on dimerization and
monomers can bind to a single `high-affinity' half-site (RGYAAC)
(Siegrist et al., 1993
;
Emery et al., 1996b
).
We hypothesize that DAF-19 is a crucial transcription factor of genes required for general cilium formation. In this case, DAF-19 recognizes symmetric X-boxes as a homodimer and strongly activates their expression in most or all CSN (Fig. 5B). If a target gene contains a more asymmetric X-box sequence, it can be recognized by heterodimers of DAF-19 together with other, as yet unidentified factors, leading then to specific expression patterns in subsets of CSN (Fig. 5B). Hypothetically, X-box motif distances from the ATG could also contribute to target gene expression variability.
DAF-19 regulates the development of a `ciliary module'
The development of sensory neurons in C. elegans is a complicated
process that includes many stages and interactions of different transcription
factors (Melkman and Sengupta,
2004). The gene daf-19 acts at late stages of sensory
neuron development when the respective cell fates have already been determined
and specification and subsequent differentiation occurs. Several different
types of genes are required to produce functional cilia in the worm: genes for
their molecular structure, genes implicated in ciliary transport machineries
and genes involved in signal reception and transduction
(Jansen et al., 1999
;
Troemel, 1999
;
Rosenbaum and Witman, 2002
;
Melkman and Sengupta,
2004
).
Our previous model associated DAF-19 regulation with only a certain group
of genes functioning in cilium morphogenesis and architecture
(Swoboda et al., 2000). The
data obtained in our current research suggest that the repertoire of
DAF-19-dependent genes is much broader
(Fig. 2). For the first time we
have found that DAF-19 can also regulate genes of ciliary function. For
example, expression of the gene odr-4 requires both a correct X-box
sequence and the presence of DAF-19. The ODR-4 protein has been shown to be an
important factor for localizing a subset of seven transmembrane domain odorant
receptors to cilia (Dwyer et al.,
1998
). Moreover, we have shown that DAF-19 can directly regulate
expression levels of some putative receptor proteins. For example, the genes
xbx-5 and xbx-6 encode a seven transmembrane domain protein
and an N-methyl-D-aspartate receptor-associated protein,
respectively.
In addition to the group of genes with signal reception properties, we
found X-boxes in promoters of proposed transcription factors
(Fig. 2). Most of these factors
contain a C2H2-type zinc-finger domain. The important role of this type of
transcription factors for the development of cell-specific properties in CSN
was already described for the gene che-1
(Uchida et al., 2003). Thus,
we predict the presence of cilium-specific developmental cascades directed by
DAF-19-dependent transcription factors. These cascades may be required for
specialized ciliary functions as well as being necessary for the possible
parallel regulation of CSN specification and their final functional
differentiation. For example, it has been demonstrated that DAF-19 can affect
the expression of indirect targets in the HOB-specific pathway through some
unknown factor(s) (the male-specific ciliated HOB neuron is necessary for
sensation of the hermaphrodite vulva during mating)
(Yu et al., 2003
). Our own
data also suggest that DAF-19 could be required not only for general cilia
formation, but in some instances for the development of cell-specific
properties as well. For example, we observed that the gene nhr-44 was
expressed in a DAF-19-dependent manner in the ciliated sensory neuron ASK.
This gene belongs to the nuclear hormone receptor family, which includes many
ligand-regulated transcriptional modulators involved in many developmental
processes (Miyabayashi et al.,
1999
).
Another example is the gene nud-1. We propose that certain
microtubule-associated molecules (like NUD-1) acting during early
developmental stages could later be recruited by DAF-19 for the purposes of
cilia development. The important role of nud-1 in nuclear migration
during embryogenesis in C. elegans was previously described
(Dawe et al., 2001). It has
also been shown that the mammalian ortholog of NUD-1, NudC, associates with
the dynein motor complex during neuronal migration
(Aumais et al., 2001
).
Therefore, we suggest a possible role for NUD-1 as a component of IFT during
ciliogenesis. In addition to nud-1, we extracted two further
X-box-containing genes from our list of candidates, spd-5
(Hamill et al., 2002
) and
dlc-1, which are also involved in nuclear migration during early
embryonic development and might later be recruited by DAF-19 for the
development of CSN.
Considering the above we propose a model where DAF-19 regulates the development of a `ciliary module' during the differentiation of sensory neurons in C. elegans (Fig. 6). According to this model, DAF-19 is a key factor for the general development of cilia. At the same time, together with other factors, it can drive the expression of genes required for specialized functions in cilia.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/8/1923/DC1
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REFERENCES |
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