Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species
(A) Comparison of the DNA sequence of the light and dark strains of ebony, as in Fig 4A. Schematics of GFP reporter constructs that pinpoint changes to the silencer of ebony are shown above and below the conservation plot. Chimeras appearing below the dark strain are drawn relative to the dark strain due to the presence of a large insertion in this strain. Similarly, chimeras above the light strain are drawn relative to the light strain due to the lack of this repetitive region. A6 midline activity was calculated using the same method described in Fig 4. The act (D) + sil (serrata) construct in (A) contains the 1kb silencer region of D. serrata placed in the context of the dark D. auraria strain ebony upstream regulatory region (Fig 6C). (B) Activity of the dark (“PM”) strain ebony upstream regulatory region, containing both activating enhancer and silencer region. Expression is specifically reduced in the dorsal midline. (C) Activity of the light (“00”) strain ebony upstream regulatory region, orthologous to that used for the dark strain. This reporter construct shows a marked increase in midline activity, recapitulating the ebony expression phenotype of this strain. (D) Chimeric reporter containing the light strain activating enhancer fused to the dark strain upstream silencer element. This reporter construct shows midline repression similar to the dark strain. (E) Chimeric reporter containing the dark strain activating enhancer fused to the light strain upstream silencer region, which recapitulates the light strain activity. (F) The CD3 truncation of the light strain reporter drives expression throughout the A6 segment, similar to the same truncation of the dark strain. (G) Graph comparing the relative activity of the Dark and Light strain Full ebony upstream reporter activity in the A4 body segment, a region unaffected by silencer activity. (H) Graph comparing the relative activity of the Dark and Light strain CD3 truncation constructs in the A4 body segment.