CHE-1 binds to the ASE motif of the wild-type cog-1 locus but not to the mutated ASE motif found in ot119 or ot201 animals. Gel shifts were done with bacterially purified CHE-1 protein as previously described (Etchberger et al. 2007). Binding to the mutated versions was assessed by cold-competition assays, using an ∼30× excess of cold wild-type or mutated probe.

Overview of the system. ASEL/R fate is controlled by a bistable feedback loop, which contains the ASEL fate inducer lsy-6, a miRNA, and the ASER fate inducer cog-1, a homeobox gene (Johnston et al. 2005). Even though both genes are asymmetrically expressed in either ASEL (lsy-6) or ASER (cog-1), both genes contain ASE motifs in their cis-regulatory regions, which are activated by the zinc-finger transcription factor CHE-1 (Etchberger et al. 2007). Cis-regulatory mutations were isolated from genetic screens in the ASE motif of lsy-6 (Sarin et al. 2007) and cog-1 (this article).

gcy-1 expression also depends on two ASE motifs. (A) Schematic of the location of the functional ASE motif in the cog-1 (Figure 3), lim-6 (Etchberger et al. 2007, 2009), and gcy-1 (B) loci. Spacings in between the 6-bp core (binding site for zinc fingers 3 and 4 of CHE-1) and translational start sites are shown. Arrows indicate the orientation of motifs. Note that we have previously documented a large number of cis-regulatory modules that drive expression in ASE and contain only a single ASE motif (e.g., gcy-5, gcy-7, lsy-6, etc.) and that completely isolated ASE motifs are sufficient to drive expression in the ASE neurons (Etchberger et al. 2007, 2009). (B) Reporter gene analysis of the gcy-1 locus. All constructs were generated by PCR fusion and scored in a otIs151 transgene background to allow identification of the ASE neurons. Mutations are complete deletions of the 12-bp site. The importance of individual ASE motifs is different, which is reminiscent of the cog-1 case, but not as drastic.

aCGH primary data. For each individual 50-mer probe, the normalized log₂ (sample fluorescence intensity/reference fluorescence intensity) is plotted at a chromosomal coordinate corresponding to the end of the oligonucleotide with the smallest coordinate, i.e., the 5′-end for probes on the plus strand and the 3′-end for probes on the minus strand. (Top two panels) The log₂ ratio for the whole region represented on the microarray but only for probes following the plus strand template. The vertical yellow lines correspond to candidate SNPs in ot119. (Bottom two panels) A small interval around the most promising candidates. The black and red circles correspond to probes designed to follow, respectively, the plus and minus strand template. The shift between the position of the minima for the plus and minus strand oligonucleotides is expected and is due to fact that on the NimbleGen platform a SNP induces a larger perturbation effect on the hybridization process when it is located close to the protruding end freely floating in the solution. The vertical dashed lines are provided to guide the eye and are reproduced in Figure 3A. More details can be found in the supplemental Materials and Methods.

Correlating ASE motif number and expression in ASE. The power of a gene's upstream ASE motifs to predict ASE expression depends on the number and score of each ASE motif. We define a gene's top-N motif score (which estimates the probability that all N motifs are functional; see materials and methods) as the product of the top-N upstream ASE motif scores for a given gene. Meanwhile we split the genes into a positive set, consisting of the 52 ASE-expressed genes [as confirmed by reporter–GFP construct experiments (Etchberger et al. 2007)] and a negative set the remainder of the genome. Then we sort the entire list of genes according to each top-N motif score, for N = 1, 2, 4, and 8 and measure how well the score criterion places the ASE-expressed genes near the top. To visualize this sorting, we generated “receiver operator characteristic” (ROC) curves. Intuitively, a ROC curve can be understood as follows: Starting at point (0, 0) at the top of the list (gene with best score), the graph moves up (y-axis) one gene if the next gene on the list is a positive and to the right (x-axis) if the gene is a negative, and so on until the last (20,183rd) gene is encountered [point (20131, 52)] at top right corner of graph. For example, point (2000, 25) on the red curve denotes that 25 ASE-expressed genes are found in the top 2025 genes (10% of the genome) in the list sorted using the top-4 motif score. Point (2000, 14) on the green curve, by contrast, shows that only 14 of the 52 ASE-expressed genes (27%) are recovered in the same-size list when sorted by the top-1 motif score (inset). Therefore, the top-4 motif score, which assumes four functional motifs and scores accordingly, is about twice as effective at identifying ASE-expressed genes than the top-1 motif score. This is statistical evidence that, on average, multiple ASE motifs are functional in ASE expression. Additionally, the underlying data from which these graphs are derived (supplemental Methods) may be interpreted as a probability estimate for ASE expression of all C. elegans genes and used to choose candidate ASE-expressed genes for further testing.

Location of ot119 and ot201 and their effect on reporter gene expression. (A) Genomic region containing the cog-1 locus. Coordinates refer to base pairs on linkage group II. See Figure 2 for explanation of the stippled interval. Conserved ASE motifs are highlighted and numbers next to the sequence indicate positions relative to the ATG start codon of the longer cog-1 isoform. The nucleotides in blue are putative transcription-factor-binding sites linked to the ASE motifs; they occur in opposite orientation and differ in relative location to each ASE motif. Shaded boxes indicate 100% conservation between all species. Black lines indicate DNA injected into the respective mutant strain to test for rescue of the ASE mutant phenotype. (B) Alignment of the cog-1 ASE motif and its mutated versions in ot109 and ot201 animals to the ASE consensus motif. “ZF” indicate the zinc fingers of CHE-1 with which it contacts its cognate binding sequence (Etchberger et al. 2007). (C) Reporter constructs. “ASE expression” indicates expression in at least one (ASER) or two ASE cells expressing gfp; note that the apparent left/right asymmetric expression of this reporter gene is brought about by transcriptional autoregulation of the translationally controlled COG-1 protein (Johnston et al. 2005). Expression was scored in young adults in a otIs151 transgene background to allow identification of the ASE neurons. In the one case in which an intermediate level of penetrance was observed (promA-del2), the brightness of the gfp signal seemed to vary in those animals where expression is observed in ASE, compared to the wild-type construct where little of such variance was observed. More details on constructs can be found in the supplemental Materials and Methods. Constructs with an * have been described in Etchberger et al. (2007) and are shown for comparison only. (D) gfp images of three animals, each expressing the indicated cog-1 reporter gene fusion and a chromosomally integrated transgene, ceh-36∷dsRed2 (otIs151), used to label ASER. Images of the green and red channel of the same animal in the same position are merged in the last set of panels. Blue arrows indicate ASER.