(A) The binding site for UNC-3 (COE motif) and LIN-39 are shown. The COE motif has been previously characterized (Kratsios et al., 2011), while the LIN-39 site was identified though ChIP-seq experiments (Boyle et al., 2014). (B–D) A schematic of the unc-129 locus is shown. Cis-regulatory mutational analysis was performed in the context of transgenic animals. Lines indicate genomic region fused to gfp (green), mCherry (red) or yfp (yellow). (+) indicates robust reporter gene expression in DA1-7/DB and head neurons. (±) indicates significant reduction in the number of neurons expressing the reporter. (–) indicates complete loss or very faint expression in the respective neurons. Multiple transgenic lines were analyzed for each construct. At least 20 animals per transgenic line were analyzed. Detailed quantification of this analysis is provided in D. Location of each COE motif (UNC-3 binding site, shown as blue vertical line) is presented as distance from ATG (+1). The blue asterisk indicates that the COE motif is conserved in at least three other nematode species. The 395 bp and the 243 bp fragments contain two well-conserved COE motifs, which are required for expression in DA1-7/DB neurons. The 395 bp fragment contains 7 LIN-39 sites (1–7, represented as black vertical lines), while the 243 bp fragment contains 3 LIN-39 sites (5-7), two of which are required for reporter gene expression in DA1-7/DB neurons. MUT indicates deletion of the LIN-39 site or that the COE motif has been mutated through substitution of 2 nucleotides in the core sequence (for example, COE wild-type site: TCCCNNGGGA >> COE MUT site: TGGCNNGGGA). Empty blue or black boxes indicate that the COE motif and LIN-39 sites are mutated, respectively. Representative images of animals carrying unc-129_243 bp::YFP and unc-129_243 bp LIN-39 sites 6 and 7 MUT::YFP are shown in C and detailed quantification is provided in D. Error bars represent standard deviation (STDV). ***p value < 0.001. N > 20 per transgenic line. (E–F) Cis-regulatory analysis of the del-1 locus. The COE3 motif in the 1827 bp fragment is required for expression in VA and VB neurons, while we have previously shown that mutation of the COE2 does not affect expression in VA and VB neurons (Kratsios et al., 2015). The 488 bp fragment is sufficient to drive reporter gene expression in VA and VB neurons. Apart from the COE3 motif, the 488 bp fragment contains 7 LIN-39 sites (1–7, represented as black vertical lines) that are predicted with p value < 0.005. Unlike the combined deletion of LIN-39 sites 5 and 6, deletion of the LIN-39 site 4 (del-1_488 bp LIN-39 site 4 MUT::YFP) results in a statistically significant loss of reporter gene expression in VA and VB neurons in two out of three transgenic lines. Detailed quantification is provided in F. Error bars represent standard deviation (STDV). ***p value < 0.0001. N > 20 per transgenic line.