Fine tuning of RFX/DAF-19-regulated target gene expression through binding to multiple sites in Caenorhabditis elegans

Abstract

In humans, mutations of a growing list of regulatory factor X (RFX) target genes have been associated with devastating genetics disease conditions including ciliopathies. However, mechanisms underlying RFX transcription factors (TFs)-mediated gene expression regulation, especially differential gene expression regulation, are largely unknown. In this study, we explore the functional significance of the co-existence of multiple X-box motifs in regulating differential gene expression in Caenorhabditis elegans. We hypothesize that the effect of multiple X-box motifs is not a simple summation of binding effect to individual X-box motifs located within a same gene. To test this hypothesis, we identified eight C. elegans genes that contain two or more X-box motifs using comparative genomics. We examined one of these genes, F25B4.2, which contains two 15-bp X-box motifs. F25B4.2 expression in ciliated neurons is driven by the proximal motif and its expression is repressed by the distal motif. Our data suggest that two X-box motifs cooperate together to regulate the expression of F25B4.2 in location and intensity. We propose that multiple X-box motifs might be required to tune specific expression level. Our identification of genes with multiple X-box motifs will also improve our understanding of RFX/DAF-19-mediated regulation in C. elegans and in other organisms including humans.

Figure 1.

(a) The location and sequence of the putative X-box motifs upstream of F25B4.2. (b) The alignment of the putative proximal and distal motifs to known X-box motifs. Also include in the alignment are the putative X-box motifs in the orthologs of F25B4.2 in three other Caenorhabditis species labeled as C. briggsae, C. remanei and C. brenneri.

Figure 2.

The alignment of F25B4.2 protein sequence with its orthologs in the other four Caenorhabditis species. CBG = C. briggsae, CRE = C. remanei, CBN = C. brenneri. The sequences were aligned using ClustalW (54) and visualized using GeneDoc (55).

Figure 3.

Genotyping of stably integrated strains. The insertion site on chromosome IV is depicted by the diagram on the top while an agarose gel showing the genotyping results on the bottom. The primers used for genotyping are also indicated by the arrows. Primer mCherry-genoF can only hybridize to inserted worms and not EG5003 and N2. The expected band sizes for inserted worms are 8312 bp from 178-genoF→ChrIV-R and 1564 bp from mCherry-genoF→ChrIV-R. The expected band size for EG5003 is 2700 bp [Mos1 is ~1280 bp (62–64)]. The expected band size for N2 is 1420 bp from 178-genoF→ChrIV-R. The gel image shows homozygous insertion for JNC20, 21, 22 and 29 as well as EG5003 and N2 as controls. The number on the right-hand side indicates the ladder positions.

Figure 4.

The expression pattern driven by dyf-5 promoter replacing the endogenous X-box motif with either the proximal motif or the distal motif. Proximal motif is able to drive normal expression while distal motif is unable to. The white arrows show the location of PHA and PHB neurons. Exposure time = 3 s.

Figure 5.

The head and tail expression patterns of F25B4.2 3 kb upstream region fused to mCherry in either wild-type (WT) strain or daf-19(m86) strain. White dashed lines outline the ciliated neurons that dye fill. Neurons that dye fill in the head include ASK, ADL, ASI, AWB, ASH and ASJ; neurons that dye fill in the tail include PHA and PHB. Because daf-19 worms do not dye fill, the white outlines are the supposed location of these neurons. The expressions in these neurons are abolished in daf-19(m86) background. The schematic of the ciliated neurons that dye fill is shown on top. The schematic is adapted from (65). Exposure time = 3 s.

Figure 6.

The expression of different deletion constructs in either wild-type or daf-19(m86) backgrounds. Proximal deletion construct removes the 15 bp putative X-box at −140; distal deletion construct removes the 15-bp putative X-box at −190; double deletion construct removes both putative X-box motifs. Other than that, all sequences remain the same. White dashed lines outline the ciliated neurons that dye fill. The outlines for strains in daf-19 background are supposed locations. (a–g) show expression in the head and (i–p) show expression in the tail. Exposure time = 3 s.