RFX transcription factors play important roles in cilia biogenesis and maintenance by transcriptionally regulate ciliary genes. Their target genes have been associated with a number of disease conditions collectively called ciliopathies.
daf-19 is the only known member of the RFX family in C. elegans. RFX regulation of ciliary genes is conserved between humans and C. elegans. The DNA binding site, known as the X-box motif, is also conserved. Thus, C. elegans has been effectively used as a model organism to identify RFX target genes. Past studies have identified target genes by searching for X-box motifs in C. elegans using bioinformatics, functional genomics, and comparative genomics methods. All of the DAF-19 target genes studied to date possess a single consensus X-box motif. Some genes (
bbs-2 and
osm-5) were found to possess two X-box motifs. We hypothesize that tandem X-box motifs could have cooperative roles. To test this hypothesis, we compared the gene sets between 4 Caenorhabditis species to find C. elegans genes with multiple X-box motifs within 500-bp promoter region. The C. elegans gene set is an extensively curated set, but this is not the case for the remaining Caenorhabditis species. In order to employ comparative genomics for X-box motif searches, we improved the gene sets for 3 Caenorhabditis species using genBlastG, a homology based gene predictor that we recently developed. Using comparative genomics with the improved gene set, we identified 15 genes that have conserved X-box motifs in all species and have multiple X-box motifs in C. elegans. We examined one gene, F25B4.2, in detail. Using singly integrated reporter constructs, we have shown that F25B4.2 is expressed in ciliated neurons. This expression is dependent on the two 15-bp X-box motifs as well as DAF-19 indicating that F25B4.2 is a DAF-19 target gene. When the proximal motif is removed, expression in ciliated neurons is ablated. When the distal motif is removed, we observed an elevated expression suggesting the distal motif has a repressive role. This is the first to report a putative repressive X-box motif in C. elegans. Our data suggest that two X-box motifs cooperate together to regulate specific expression level of F25B4.2. We model that having multiple X-box motifs in the promoter could achieve specific expression level. Our identifications of X-box motifs will improve our understanding on RFX mediated regulation in C. elegans and in other organisms including humans.