To better understand how enhancers are built, we studied independently evolved enhancers active at a similar time and space in fruit flies. These enhancers each regulate the expression of the gene yellow in the wing, a gene necessary for black pigment production. The developmental expression pattern of yellow prefigures the black pigmentation in the adult. In three different species (D. biarmipes, D. tristis and D. nebulosa), a black spot has been independently gained at the tip of the wing, and this is partly due to the independent acquisition of ‘spot’ enhancers of yellow.
Our lab focused on the ‘spot’ enhancers of the ‘spotted’ species D. biarmipes and D. tristis. Benjamin Prud’homme’s lab in Marseille concentrated on D. nebulosa ‘spot’enhancer. We aimed at determining the precise location of the ‘spot’ enhancers in the 3 species. This showed that in each case, the ‘spot’ enhancers are overlapping (or in close vicinity) with older enhancers, present as well in non-spotted species.
We also aimed at finding which transcription factors bind these ‘spot’ enhancers. Our approach was a RNAi screen for the 3 ‘spot’ enhancers, where we blocked the expression of transcription factors, one at a time, and checked whether the ‘spot’ enhancer still functioned normally. If the function of one enhancer was impaired in the absence of a given transcription factor, we concluded that this transcription factor was probably involved in the regulation of the ‘spot’ enhancer. We obtained a list of candidate regulators for each ‘spot’ enhancer, and realized that some regulators were the same for all 3 enhancers.
To confirm the importance of these regulators, we mutated putative target motifs of these transcription factors in each enhancer. The mutated enhancers had an impaired function, suggesting that the candidate transcription factors may regulate the enhancers directly.
For one of these candidate transcription factors, the project will continue, testing the directeness of the transcription factor binding, using a technique called ChIP-seq. The principle is to ‘freeze’ all interactions in the tissue of interest (here the wing of each of the 3 species) by crosslinking, and then to select the fragments of DNA bound by the transcription factor of interest by immunoprecipitation. These fragments are then sequenced to reveal where the factor binds on the DNA, and in particular whether it is present at the level of the ‘spot’ enhancer of each species.
The results of this project have been presented to non-scientists gradually every year at the Open day of the Biocenter (Ludwig-Maximilians Universität, Munich). Once the results of the ChIP-seq experiment are known, the collaborative work will be published in a scientific journal in open access.
