Thesis defense: Margarita Masoura

On December 4th, Margarita Masoura of the team of Yad Ghavi-Helm will support her thesis entitled:

"Pleiotropy, Specificity, and Sequence Dissection of the E3 Enhancer of twist during Drosophila Embryogenesis"

Abstract:

Precise gene expression patterns during Drosophila embryogenesis arise through the action of non-coding DNA elements called enhancers. These elements activate transcription by interacting with gene promoters of their targets -even when separated by large genomic distances- through 3D chromatin looping. Traditionally, enhancers are viewed as modular and tissue-specific elements, each acting in a single well-defined spatiotemporal context. However, accumulating evidence pinpoints the prevalence of pleiotropic enhancers -sequences that activate one gene in multiple contexts- raising questions on how such elements can function. Pleiotropic enhancers usually contain high amount of regulatory information and confer broad activity, which is often reflected in the large number of Transcription Factor Binding Sites (TFBSs) embedded in their sequences. Given that wide regulatory potential it becomes conceivable to hypothesize that they might regulate multiple genes. While this idea has been previously suggested, it remains completely unexplored. Moreover, how such broad regulatory capacity is encoded in their sequence architecture also remains unclear.

In my thesis, I address these questions by studying the E3 enhancer, previously known to regulate twist -the master regulator of mesodermal fate- during early and mid-embryogenesis of Drosophila melanogaster. I first show that E3 is pleiotropic, driving expression across germ layers and developmental stages, including tissues where twist is not expressed. I then demonstrate that E3 regulates at least three additional, functionally unrelated genes. Despite E3’s broad activity, each target gene maintains a specific, non-overlapping expression pattern. This specificity arises from promoter-proximal elements, which interpret E3’s broad regulatory input and refine it into precise transcriptional outputs.

Regarding the sequence organisation of E3, I demonstrate that the pleiotropic nature of the enhancer is encoded in three core domains, each driving expression in a discrete spatiotemporal context. Despite this modularity, regulatory sites that fine-tune expression levels in one context can lie within domains that specify another context. Targeted and saturating mutagenesis on E3 sequence confirm this intermingling of regulatory sites, showing that, at least for E3’s mesodermal activity, transcriptional output arises from the combinatorial action of regions distributed across the entire enhancer.

Altogether, my thesis shows how a pleiotropic enhancer can generate broad regulatory outputs while individual gene expression specificity is maintained by promoter-proximal elements. It also demonstrates that enhancer activity relies on multiple regulatory regions spread across the genome, that act in concert to produce precise transcriptional patterns.