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. 2018 Mar 22;7:e32785. doi: 10.7554/eLife.32785

Figure 5. HSN transcription factor combination acts directly on target genes.

(A) tph-1 minimal HSN CRM (tph-1prom2) mutational analysis. Black crosses represent point mutations to disrupt the corresponding TFBS. +: > 60% of mean wild type construct values; +/−: expression values 60–20% lower than mean wild type expression values; −: values are less than 20% of mean wild type values. n > 60 cells per line. x/y represents the number of lines with the expression pattern (x) from the total lines analyzed (y). See Figure 5—figure supplement 1 for raw values and nature of the mutations and Figure 5—figure supplement 2 for in vitro binding. (B) tph-1prom2::gfp expression is partially affected in egl-18(ok290) mutants. In red, significant defects relative to wild type. n > 100 cells for each genotype. (C) cat-1 minimal HSN CRM (cat-1prom14) mutational analysis. (D) cat-1prom14::gfp expression is unaffected in egl-46 mutants, which coincides with the lack of phenotype when INSM binding sites are mutated in this construct. cat-1prom14::gfp contains functional GATA sites and, as expected, its expression is affected in egl-18 mutants. Expression of a longer reporter (cat-1prom1::gfp) is independent of egl-18 revealing compensatory effects in the context of big regulatory sequences. (E) bas-1 minimal HSN CRM (bas-1prom18) mutational analysis. (F) A longer bas-1 construct (bas-1prom13) is more robustly expressed in HSN (90% expression compared to mean 48% expression of bas-1prom18 reporter lines). This construct contains functional INSM binding sites. (G) bas-1prom18::gfp expression is affected in ast-1(ot417) and egl-18(ok290) mutants. Expression of a longer reporter (bas-1::prom1) is independent of ast-1 and egl-18 revealing compensatory effects in the context of big regulatory sequences. (H) GATA-binding site point mutation does not significantly affect bas-1::gfp expression in the wild type background (no significant difference between mean expression of three lines of bas1prom1 and three lines of bas1prom18). However, it synergizes with ast-1 mutant background leading to a complete loss of GFP expression. These results unravel a direct role for GATA sites in bas-1 gene expression and synergy between egl-18 and ast-1.

Figure 5.

Figure 5—figure supplement 1. Primary data from the mutagenesis analysis (Figure 5).

Figure 5—figure supplement 1.

Each number represents the % of GFP cells in a particular transgenic line. +: > 60% of mean wild type construct expression, +/−: values indicate a penetrance of 20–60% the mean wild type expression value; −: values are less than 20% of mean wild type values. n > 60 cells per line. Above each construct, the wild type consensus sequences are included in capital letters in a longer region context and after the arrow the point mutations are highlighted in red. Related to Figure 5.
Figure 5—figure supplement 2. UNC-86, EGL-18 and AST-1 bind to the 5HT pathway gene CRMs in electrophoretic mobility assays.

Figure 5—figure supplement 2.

(A) Purified UNC-86 binds tph-1, cat-1 and bas-1 CRMs in a concentration dependent manner (depicted by arrowheads). UNC-86 binding is aboDlished by point mutation in the POU-binding site (mut lanes). (B) Similarly, purified AST-1 binds to cat-1 and bas-1 CRMs in a concentration-dependent manner (arrowheads). AST-1 binding is lost upon ETS-binding site mutation (mut lanes). (C) Cellular extracts from HEK293T cells overexpressing EGL-18:HIS bind the cat-1 CRM. HIS antibody but not GFP antibody causes a shift in EGL-18:HIS band (compare Shift to Supershift bands) indicating that binding involves EGL-18 protein. Moreover, point mutation of GATA site abolishes cat-1 sequence binding by the cellular extract (mut lanes).