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Abstract

During development, complex gene expression patterns are formed, relying on the spatiotemporal coordination of proteins and genomic regulatory regions, among many other players and layers of processes. Low-affinity binding sites efficiently contribute to transcription through brief interactions with transcription factors. Concentrating transcription factors in localized environments, driven by clusters of enhancers, have been suggested as a mechanism by which low-affinity sites are efficiently used. Such localized transcriptional environments have been observed in Drosophila melanogaster embryos for the transcription factor Ultrabithorax (Ubx) (Tsai et al., 2017) at the locus of its target shavenbaby (svb). In this thesis, I interrogate the complex role of Ubx and multi-enhancer transcriptional hubs throughout development. Firstly, I showed that active svb enhancers on different chromosomes tend to co-localize. I then tested the hypothesis that multi-enhancer interactions contribute to low-affinity transcriptional microenvironments and show that defects from a deletion of a redundant enhancer from the svb locus at elevated temperatures can be rescued by introducing svb’s cis-regulatory sequence driving a reporter in a different chromosome, which suggests that multiple enhancers can reinforce local transcriptional hubs to buffer against environmental stresses. Secondly, I explored whether microenvironments are specific to low-affinity enhancers or if microenvironments can form around Ubx high-affinity enhancers. I found that Ubx local enrichment is overall maintained when low-affinity sites in a svb enhancer are substituted with high-affinity sites. Then, I screened a library of short genomic fragments containing either (endogenous) Ubx high-affinity sites or mutations of these to assay their expression patterns and transcriptional microenvironment features. I show that sequences containing high-affinity sites can function as transcriptional enhancers across development and can exhibit features of multi-enhancer transcriptional microenvironments such as Ubx local enrichment and transcript co-localization with Ubx target svb. Finally, it remained to be tested what is the extent of the contribution of Ubx to these phenomena, so I developed a Ubx recruitment system to explore the role of this transcription factor in enhancer clustering using high-resolution microscopy. Overall, this project sheds new light on transcriptional microenvironments, providing a more in-depth understanding of their components, exploring them in the context of gene regulation during animal development.