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 (called E3) which regulates the twist gene in the Drosophila embryo.
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 enhancer-reporter assays, we were able to show that the spatio-temporal activity of this enhancer is different from the activity of the gene it regulates, opening interesting new avenues of research.
Following up on this initial result, we discovered that the E3 enhancer is pleiotropic. It does not only regulate the expression of twist, but is shared between at least 3 different genes.
Interestingly, for one of these genes, E3 drives expression in a tissue of ectodermal origin, which contrasts with twist being a mesodermal transcription factor.
Overall, we have shown that E3 is a pleiotropic enhancer that functions across germ layers and regulates the expression of multiple genes even across large genomic distances. Tissue specificity is driven by the specific sequence of each gene's promoter.
These results are currently been written up in a manuscript in preparation.
In another part of this sub-aim, we reintroduced our enhancer of interest at various locations in the genome in a context where the endogenous E3 enhancer was deleted.
Surprisingly, one of the insertion sites could rescue the lethality of the deletion although the ectopic enhancer was introduced 51kb away across a TAD boundary.
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 two of the above-mentioned lines, despite a very large distance separating them.
Bioinformatics analysis confirmed that a lot of enhancer-promoter interactions occur across TAD boundaries in the Drosophila genome. We have proven their functionality in at least one case.
These results were published in Nucleic Acids Research (Balasubramanian et al.).
We are currently following up on this work by extending the number of functional enhancer-promoter interactions to decipher the drivers of long-range inter-TAD enhancer-promoter interactions.
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 compared to the wild-type using qRT-PCR and immunostaining.
We also developed a high-throughput method to analyze their effect on gene expression by adapting the HCR-flowFISH method to Drosophila embryos.
We are currently analyzing the data.
WP3: Large-scale enhancer activity screen
We have developed a novel in vivo / in silico method for spatial single-cell enhancer-reporter assays (spatial-scERA) designed to reconstruct the spatial activity of candidate enhancer regions in parallel in multicellular organisms.
Spatial-scERA integrates parallel reporter assays with single-cell RNA sequencing and spatial reconstruction using optimal transport, to map cell-type-specific enhancer activity at the single-cell level on a 3D virtual sample. We evaluated spatial-scERA in Drosophila embryos using 25 candidate enhancers, and validated the robustness of our reconstructions by comparing them to in situ hybridization. Remarkably, spatial-scERA faithfully reconstructed the spatial activity of these enhancers, even when the reporter construct was expressed in as few as 10 cells. Our results demonstrate the importance of integrating transcriptomic and spatial data for accurately predicting enhancer activity patterns in complex multicellular samples and linking enhancers to their potential target genes. Overall, spatial-scERA provides a scalable approach to map spatio-temporal enhancer activity at single-cell resolution without the need for imaging or a priori knowledge of embryology and can be applied to any multicellular organism amenable to transgenesis.
These results have been published as a preprint (Alberti et al) and have been accepted upon minor revision at Nucleic Acids Research.
