Redundant and Cryptic Enhancer Activities of theDrosophila yellowGene

Abstract

Cis-regulatory sequences known as enhancers play a key role in regulating gene expression. Evolutionary changes in these DNA sequences contribute to phenotypic evolution. The Drosophila yellow gene, which is required for pigmentation, has emerged as a model system for understanding how cis-regulatory sequences evolve, providing some of the most detailed insights available into how activities of orthologous enhancers have diverged between species. Here, we examine the evolution of yellow cis-regulatory sequences on a broader scale, by comparing the distribution and function of yellow enhancer activities throughout the 59 intergenic and intronic sequences of Drosophila melanogaster, D. pseudoobscura, and D. willistoni. We find that cis-regulatory sequences driving expression in a particular tissue are not as modular as previously described, but rather have many redundant and cryptic enhancer activities distributed throughout the regions surveyed. Interestingly, cryptic enhancer activities of sequences from one species often drove patterns of expression observed in other species, suggesting that the frequent evolutionary changes in yellow expression observed among Drosophila species may be facilitated by gaining and losing repression of preexisting cis-regulatory sequences.KEYWORDS cis-regulatory element; evolution; pigmentation; novelty; gene expression C IS-REGULATORY elements known as enhancers affect development and physiology by controlling the time, place, and amount of mRNA that is transcribed from a gene. These DNA sequences range from hundreds to thousands of base pairs, are typically located in noncoding regions of the genome, and contain binding sites for transcription factors (Spitz and Furlong 2012;Long et al. 2016). In multicellular organisms such as Drosophila, most genes are regulated by multiple enhancers. Early studies suggested that each enhancer was responsible for a unique subset of a gene's expression (e.g., Davidson 2001), but it is now known that multiple enhancers with varying degrees of functional redundancy can also contribute to the expression of a gene in a given cell

Gene Regulatory Network Homoplasy Underlies Recurrent Sexually Dimorphic Fruit Fly Pigmentation

“…in isolated reporter constructs (Kalay et al, 2019). Thus, the repeated GRN inclusion of yellow, tan, and ebony may indeed be due to convergence at the level of its CREs, a hypothesis that can now be tested more rigorously.…”

A silencer repressing redundant enhancer activities revealed by deleting endogenouscis-regulatory element ofebonyinDrosophila melanogaster

“…Therefore, redundant enhancer elements are likely to be located elsewhere. However, unlike the recent reporter assay conducted with the yellow regulatory region [23], many regions were tested using relatively large fragments (> 2 kbp), which may contain cryptic enhancers that are repressed by surrounding sequences in their native genomic context. Therefore, the possibility of the presence of redundant enhancer elements within the approximately 10 kbp regulatory region cannot be ruled out.…”

“…This long-range repression eliminates the need for an acquisition of repressor-binding sites for individual enhancer elements. A study of yellow , which is also expressed in the developing epidermis, from three different species revealed a contrasting picture of frequent evolutionary acquisition and loss of short-range repressor binding sequences [23]. Also, reporter assays examining the effect of the male-specific silencer of ebony in D. auraria and D. serrata observed a frequent loss of this silencer in these species [53].…”

“…For example, the distinct CREs of yellow that activate transcription in bristles, wing and body, and abdomen have been identified [18–22]. However, a recent study revealed that many sequence fragments in the regulatory region of yellow exhibit redundant and cryptic enhancer activities, suggesting that cis -regulatory modules are not as distinct as described previously and more amenable to evolutionary changes [23].…”

“…However, some limitations exist because this assay does not test the sequence fragments in their native genomic environment [36]. Especially, the lengths and borders of the sequence fragments can markedly affect the results [23]. In this study, rather than conducting reporter gene assays, we examined the genomic region by modifying the endogenous upstream sequence using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system.…”