Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Spotifly (Genetic paths underlying the convergent evolution of pigmented spots on fly wings)

Teaser

In all animals, genes are only expressed (~active) in a subset of cells and each cell type is determined by a specific combination of expressed genes. The expression of genes is thus regulated, and this regulation depends on sequences controlling “where” and “when”...

Summary

In all animals, genes are only expressed (~active) in a subset of cells and each cell type is determined by a specific combination of expressed genes. The expression of genes is thus regulated, and this regulation depends on sequences controlling “where” and “when” genes are expressed, the ‘enhancers’. Enhancers, when bound by specific transcription factors, stimulate the expression of their target gene. Transcription factor binding depends on specific sequence motifs. The mere introducing of these motifs in any sequence, however, doesn’t result in a functional enhancer. There must be rules, still unclear, that govern the functionality of enhancers. Understanding these rules is critical, as all biological processes depend on the correct expression of specific genes in space and time.

Deciphering these rules might be made easier by comparing enhancers with a similar function that have evolved independently. This comparison would reveal what is constrained and what is flexible in the organization of an enhancer. In collaboration with Benjamin Prud’homme’s lab in Marseille, we studied 3 independently evolved enhancers active in the anterior part of the wing in 3 fruit fly species. While hundreds of transcription factors are present in this tissue, we found three that regulate at least 2 of these enhancers, suggesting strong biases in the evolutionary recruitment of these factors. We also found that these 3 enhancers evolved in the vicinity of (different) pre-existing enhancers, suggesting that the evolutionary path to a novel enhancer may be shortened by the re-use of some of the features of an “older” enhancer.

Work performed

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.

Final results

The results of this project suggest two interesting ideas.
First new enhancers probably don’t evolve randomly anywhere in the DNA sequence, but tend to cluster with pre-existing enhancers. This opens new questions. What do the new enhancers share with the older ones? Some binding sites for transcription factors? Accessibility of the DNA?
Second, to generate a new enhancer function, even if hundreds of transcription factors are theoretically available, it seems that some are chosen preferentially. The reason for such biases is also unclear. Are the binding sites for those factors easier to gain (fewer mutations required)?
This project participates in deepening our understanding of enhancers. This knowledge is critical, as mutations in enhancers are known to be involved in some human genetic disorders (like X-linked deafness or pre-axial polydactyly) and in cancer.

Website & more info

More info: http://www.evolutionary-ecology.bio.lmu.de/evolution_development/index.html.