DEV030064 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. gfp expression patterns for deletion analysis of gcy-5, gcy-7, lim-6 and lsy-6 cis-regulatory sequences. Examples of gfp expression observed from deletion analysis constructs. For gcy-7, gcy-5 and lsy-6 some constructs show no phenotype while others ectopically express gfp in the contralateral cell (indicated by yellow arrowhead). Multiple lim-6 deletions exhibit loss of gfp expression in ASEL (location of ASEL indicated by yellow arrowhead).

• Supplemental Figure S2 -

  Fig. S2. Quantification of gcy-5, gcy-7, lim-6 and lsy-6 scanning deletion analysis. Construct information can be provided on request. Expression of gfp in ASEL/R was assessed with otIs151 (Isceh-36::DsRed2) in the background. Multiple independent transgenic lines were analyzed for each reporter construct. One representative line is shown for each reporter.

• Supplemental Figure S3 -

  Fig. S3. Orthologous alignments of gcy-7 and lim-6 cis-regulatory sequences. Sequence alignments from C. brenneri, C. briggsae, C. remanei and C. elegans. All sequences end exactly before the predicted ATG translational start site of the respective gene. Complete conservation is indicated by an asterisk (*) below the sequence. ASE motifs are highlighted in blue. Black and red lines above the sequence indicate the location of deletions from Fig. 4. Red lines indicate that the deletion had an effect on gfp expression. Black lines indicate no effect. The gcy-5 promoter sequence cannot be aligned between nematodes and the C.briggsae gcy-5 promoter is also not expressed in ASER (data not shown). lsy-6 alignments are shown in Fig. 4D.

• Supplemental Figure S4 -

  Fig. S4. CEH-36 binds to a functionally relevant motif in the gcy-7 promoter. (A) Sequence of the ASE^gcy-7#2 regulatory motif that drives L/R asymmetric expression (ASEL>ASER). The sequence is conserved in C. briggsae and the C. briggsae gcy-7 promoter is indeed expressed in ASEL when injected in C. elegans (data not shown). (B) EMSAs for CEH-36 binding to probe alone (left panel) and together with CHE-1 (right panel). CEH-36 binds to the TAATCC sequence as shown by cold competition with mutA but not mutB. CEH-36 binding occurs in parallel and independent of the binding of CHE-1 to the neighboring ASE motif. CEH-36 can bind the regulatory element in the absence of CHE-1 protein and in the absence of a functional ASE motif. CEH-36 can also supershift a CHE-1 complex bound to an oligonucleotide containing the ASE motif and the adjacent TAATCC motif. The Prd-type homeodomain protein CEH-10 and the LIM-homeodomain protein TTX-3 are not able to bind the motif or to supershift the CHE-1/DNA complex (see Fig. 5). For all lanes, the labeled probe was the wild-type sequence.

• Supplemental Table S1 - Adobe PDF