AbstractThe spatiotemporal regulation of gene expression is essential to ensure robust phenotypic outcomes. Pigmentation patterns inDrosophilaare formed by the deposition of different pigments synthesized in the developing epidermis and the role ofcis-regulatory elements (CREs) of melanin biosynthesis pathway-related genes is well-characterized. These CREs typically exhibit modular arrangement in the regulatory region of the gene with each enhancer regulating a specific spatiotemporal expression of the gene. However, recent studies have suggested that multiple enhancers of a number of developmental genes as well as those ofyellow(involved in dark pigment synthesis) exhibit redundant activities. Here we report the redundant enhancer activities in thecis-regulatory region of another gene in the melanin biosynthesis pathway,ebony, in the developing epidermis ofDrosophila melanogaster. The evidence was obtained by introducing an approximately 1 kbp deletion at the endogenous primary epidermis enhancer (priEE) by genome editing. The effect of the priEE deletion on pigmentation and on the endogenous expression pattern of amCherry-taggedebonyallele was examined in the thoracic and abdominal segments. The expression level ofebonyin the priEE-deleted strains was similar to that of the control strain, indicating the presence of redundant enhancer activities that drive the broad expression ofebonyin the developing epidermis. Additionally, the priEE fragment contained a silencer that suppressesebonyexpression in the dorsal midline of the abdominal tergites, which is necessary for the development of the subgenusSophophora-specific dark pigmentation patterns along the midline. The endogenous expression pattern ofebonyin the priEE-deleted strains and the reporter assay examining the autonomous activity of the priEE fragment indicated that the silencer is involved in repressing the activities of both proximal and distant enhancers. These results suggest that multiple silencers are dispensable in the regulatory system of a relatively stable taxonomic character. The prevalence of other redundant enhancers and silencers in the genome can be investigated using a similar approach.Author summaryGenes are expressed at the right timing and place to give rise to diverse phenotypes. The spatiotemporal regulation is usually achieved through the coordinated activities of transcription-activating and transcription-repressing proteins that bind to the DNA sequences called enhancers and silencers, respectively, located near the target gene. Most studies identified the locations of enhancers by examining the ability of the sequence fragments to regulate the expression of fused reporters. Various short enhancers have been identified using this approach. This study employed an alternative approach in which the previously identified enhancer that regulates expression ofebony(a gene involved in body color formation) was deleted in a fruitfly,Drosophila melanogaster, using the genome-editing technique. The knockout of this enhancer did not affect the transcription level of the gene to a large extent. This indicated the presence of transcription-activating elements with redundant functions outside the deleted enhancer. Additionally, the transcription ofebonyat the midline of the abdomen, which is repressed in the normal flies, were derepressed in the enhancer-deleted flies, which indicated that the deleted enhancer fragment contained a silencer that negatively regulates multiple enhancer activities in a spatially restricted manner.
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