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Regulatory genomics during Drosophila embryogenesis: dissecting enhancer-promoter interactions

Periodic Reporting for period 2 - Enhancer3D (Regulatory genomics during Drosophila embryogenesis: dissecting enhancer-promoter interactions)

Reporting period: 2019-11-01 to 2021-04-30

In eukaryotes, the complex regulation of temporal- and tissue-specific gene expression is controlled by the binding of transcription factors to enhancers, which in turn interact with the promoter of their target gene(s) via the formation of a chromatin loop. Despite their importance, the properties governing enhancer function and enhancer-promoter loops in the context of the three-dimensional organisation of the genome are still poorly understood.
My recent work suggests that (i) developmental genes are often regulated by multiple enhancers, sometimes located at great linear distances, (ii) the spatio-temporal activity of a large fraction of those enhancers remains unknown, (iii) enhancer-promoter interactions are usually established before the target gene is expressed and are largely stable during embryogenesis, and (iv) stable interactions seem to be associated with the presence of paused RNA Polymerase II at the promoter before gene activation.
Building upon these results, we propose to advance to the next level in the dissection of enhancer-promoter interaction functionality in the context of Drosophila embryogenesis. Specifically, we will address three important questions:
(WP1) What determines the specificity of promoter-enhancer interactions in a complex genome?
(WP2) Are enhancer-promoter interactions tissue-specific, and what are the drivers of this specificity?
(WP3) Are all enhancer-promoter interactions functional, and how does the activity of an enhancer relate to the expression of the gene it interacts with?
To this end, my group combines state-of-the-art methods in genetics and genomics, including novel single-cell techniques, using Drosophila embryogenesis as a model system. Our results will provide a unique view of the functionality of enhancer-promoter interactions in a developing embryo, a significant step towards understanding the link between chromatin organisation and transcription regulation.
WP1: Specificity of promoter-enhancer interactions in a complex genome
We have generated a number of mutant fly lines to study the role of our enhancer of interest.
Sub-aim 1a:
In a first project, we started by deleting this enhancer. Surprisingly, the deletion is lethal, which was not expected based on previous knowledge about this enhancer. Using RT-qPCR and immunostaining, we were able to show that the temporal activity of this enhancer is different from the activity of the gene it regulates, opening interesting new avenues of research.
We then reintroduced our enhancer of interest at various locations in the genome. Surprisingly, one of the insertion sites could rescue the lethality of the deletion. We then analyzed the effect of these mutants on chromatin organization (by 4C-seq and 3D DNA FISH) and found that the ectopic enhancer can interact with the promoter of its target gene in the two above-mentioned lines, despite a very large distance separating them.
Sub-aim 1-b:
In a second project, we generated a number of mutants targetting important motifs in our enhancer on interests. We analyzed the effect of these mutants in comparison to the wild-type using qRT-PCR and immunostaining.

WP3: Large-scale enhancer activity screen
We have developed a new method to assess enhancer activity using single-cell RNA-seq instead of imaging.
We spent a lot of time optimizing the method and in particular choosing the best reporter gene.
We have generated 4 enhancer-reporter constructs and used them as a proof of concept of the method.
A first set of single-seq RNAseq data was generated and is currently been analyzed.
We have studied a developmental gene and its enhancers in great detail. The results suggest a new mode of regulation, and we currently following up on these interesting results.
The results of sub-aim 1a suggest that, at least in Drosophila, the distance between an enhancer and a promoter, and the fact that they are in the same topological domain, might be less important than expected for gene expression. We are exploring other factors that might explain why enhancers and promoters can interact across large distances.
Finally, we have developed a new method to assay enhancer activity at high throughput. We are currently applying it to a large set of previously-uncharacterized enhancer.