To identify the position, number, kind, and size of effects of functionally relevant mutations within the ebony abdominal enhancer, we compared dark U76 and light U62 alleles because these represent the two extremes of ebony expression. Between the U76 and the U62 alleles, there are ~120 nucleotide differences scattered over the 2.4-kb abdominal enhancer [44 point mutations and 76 base pairs (bp) differing because of 10 insertions or deletions (indels)] that could potentially contribute to the observed difference in activity. To localize functional differences, we first created chimeric reporter constructs with groups of mutations and then narrowed these to individual changes that contribute to the activity of chimeric constructs. Our analysis below suggests that a minimum of five mutations differentiate the activities of dark and light lines, two of which are specific to the dark haplotype.
We focused on a 2.4-kb region that contained the 0.7-kb core abdominal element (“abd” in ) and recapitulated the difference in RNA expression between dark (U76) and light (U62) lines, such that the dark allele construct expressed 22% of the reporter activity of the light allele construct (fig. S8, B and C
). We subdivided the 2.4-kb region into three subregions (X, Y, and Z, ) and systematically substituted individual fragments from the light allele into the dark allele construct. Of the three subregions tested, the Z fragment showed the strongest effect (fig. S8D
), increasing reporter activity from 22% to 67% of the activity of the light allele. Moreover, in the reciprocal construct, swapping in the Z fragment was sufficient to decrease activity of the light allele from 100% to 46% activity (fig. S8E
Fig. 5 Multiple mutations in the ebony abdominal cis regulatory element contribute to enhancer activity differences. Five mutations decrease ebony expression in the dark allele and show a varied distribution across Africa. (A) Schematic of ebony abdominal enhancer, (more ...)
Several previously identified candidate mutations were only observed on the dark haplotype, the majority of which (five out of eight) map to the 2.4-kb regulatory region (fig. S8A
, red bars labeled “Dark Specific Substitutions”). Replacement of all five substitutions in the dark allele construct with the nucleotides present in the light allele increased reporter activity to 70% of that of the light allele construct, demonstrating that they include functionally important mutations (fig. S8F
). The Z fragment contains four of the five dark-specific mutations within the 2.4-kb element (fig. S8A
), so we reverted the individual dark-specific substitutions of the dark allele construct. Dark-specific substitutions 2 and 3 showed no effect on the level of reporter expression (table S2
), whereas substitution 4 showed a small effect on expression, raising activity from 22% to 35% (fig. S8G
). Substitution 5, however, caused a dramatic increase to 64% of the light allele activity (fig. S8H
). Therefore, at least two novel substitutions in the Z fragment have contributed to the divergence of the dark and light haplotypes, with substitution 5 providing the largest effect.
To account for the remaining ~50% difference in activity, we turned to the X and Y fragments. No contribution of the X fragment (which contained dark-specific substitution 1) was observed when the light allele X fragment was swapped into the dark allele construct (fig. S8I
). However, the chimeric construct bearing the Y fragment from the light line increased activity from 22% to 47% (fig. S8J
), demonstrating that one or more functional mutations exist in the Y fragment. The Y fragment encompasses the core abdominal activity, the smallest span of DNA sufficient to drive strong reporter expression in the abdomen (figs. S6 and S8A
). Comparison of the isolated Y fragment activities of the light and dark alleles also revealed much weaker activity of the dark allele Y fragment construct (25% relative to light) (fig. S9, B and C
In order to pinpoint causative mutations within the Y fragment, we assayed a series of Y fragment GFP reporter constructs. Twenty-five point mutations and four indels (encompassing 42 bp) exist between the light U62 and dark U76 alleles Y fragment sequences. The major contribution to expression differences (81%) mapped to the 5′ half of the Y fragment (fig. S9, D and E
), which allowed us to narrow the 67 candidate nucleotides down to the eight point mutations that differ in this region of the Y fragment (). Of these eight candidates, three were eliminated because they were found in other strongly expressing Y fragments. We reverted each of the five remaining candidate substitutions individually from the dark allele to that present in the light allele. Mutations at positions 27 and 32 increased dark allele Y fragment activity from a 25% baseline to 54% and 50%, respectively, of the light allele Y fragment activity (fig. S9, F and G
). The third substitution at position 137 had a more dramatic effect on the Y fragment activity, raising expression to 80% of light Y fragment expression (fig. S9H
) (note that the sum of effects exceeds 100%, so individual effects are not strictly additive). These results suggest that at least three substitutions within the Y fragment contributed to the overall reduction of abdominal enhancer activity.
The five mutations that functionally differentiate the dark and the light haplotypes cause a decrease in the activity of the dark allele enhancer. The mutation with the greatest effect arose at a considerable distance (270 bp) from the core element. If this mutation is in an activator binding site, we would expect that this sequence would lie in the core element. Alternatively, the mutation may represent a repressor binding site. Indeed, when we deleted this site and the five adjacent nucleotides 5′ and 3′ to it, the enhancer drove a dramatic increase in reporter expression, from 22% to 106% of the light haplotype activities (fig. S8L
). The greater effect of deleting these sites relative to reverting the nucleotide raised the possibility that these sites serve a function in the light allele. When we engineered the identical deletion into the light haplotype, reporter activity also increased (fig. S8M
, 170% of light haplotype), indicating that this sequence is required to repress enhancer activity and that the substitution in the dark haplotype further repressed ebony
Together, these data show that multiple mutations (at least five), with varying effects (accounting for 8% to 40% of the overall difference in activity) and representing different kinds of functional change (reduced activation strength, increased repression), underlie the evolution of the ebony abdominal enhancer.