Large-scale analysis ofDrosophilacore promoter function using synthetic promoters

Abstract

The core promoter, the region immediately surrounding the transcription start site, plays a central role in setting metazoan gene expression levels, but how exactly it computes expression remains poorly understood. To dissect core promoter function, we carried out a comprehensive structure-function analysis to measure synthetic promoters activities, with and without an external stimulus (hormonal activation). By using robotics and a dual-luciferase reporter assay, we tested ~3000 mutational variants representing 19 different Drosophila melanogaster promoter architectures. We explored the impact of different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, positioning, and flanking sequences. We observe strong effects of the mutations on activity, and a linear combination of the individual motif features can largely account for the combinatorial effects on core promoter activity. Our findings shed new light on the quantitative assessment of gene expression, a fundamental process in all metazoans.

Targeted mutagenesis of specific genomic DNA sequences in animals for thein vivogeneration of variant libraries

“…The advent of MPRAs has revolutionized our ability to query biological phenomena, from dissecting gene regulatory regions to systematically studying protein function (Arnold et al, 2013; Brown et al, 2022; Chan et al, 2023; Inoue and Ahituv, 2015; Ireland et al, 2020; Jores et al, 2020; Kheradpour et al, 2013; Lagunas et al, 2023; Lalanne et al, 2024; Qi et al, 2020; Staller et al, 2022). Yet, these advances have been mostly relegated to cells in culture, where the transfection of massive libraries of DNA is feasible.…”

“…To uncover these rules it is therefore necessary to find the binding sites within an enhancer with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively Parallel Reporter Assays (MPRAs) have made it possible to perform this feat in the context of cells in culture and in some multicellular organisms (Arnold et al, 2013; Brown et al, 2022; Chan et al, 2023; Inoue and Ahituv, 2015; Ireland et al, 2020; Jores et al, 2020; Kheradpour et al, 2013; Lagunas et al, 2023; Lalanne et al, 2024; Qi et al, 2020). Here, a barcoded library of reporter genes, each driven by a randomly mutated regulatory region or genome fragments, is generated in vitro and transfected into cells.…”

“…These correlations yield an information footprint that reveals activator and repressor binding sites (Ireland et al, 2020). Further, by computationally designing these libraries, regulatory parameters such as the relative placement of binding sites can be systematically tested (de Almeida et al, 2022; Kircher et al, 2019; Qi et al, 2020; Sharon et al, 2012; Yu et al, 2021). The result has been a pipeline that allows for the rapid identification and experimental validation of binding sites within an enhancer, and for the engagement in a theory-experiment dialogue aimed at reaching a predictive understanding of how binding site architecture dictates gene expression.…”

“…A second limitation of MPRAs-which applies to both cells in culture and multicellular organisms-is their reliance on reporter constructs: enhancers within the library are typically integrated into a plasmid that allows for the simultaneous measurement of enhancer sequence and reporter gene expression level (Arnold et al, 2013;Brown et al, 2022Brown et al, , 2022Chan et al, 2023Chan et al, , 2023Farley et al, 2015;Inoue and Ahituv, 2015;Ireland et al, 2020;Jores et al, 2020Jores et al, , 2020Kheradpour et al, 2013;Lagunas et al, 2023Lagunas et al, , 2023Lalanne et al, 2024;Qi et al, 2020). In most cases these reporter constructs remain episomal and lack chromatin such that they cannot capture information about the genomic context that might be at play such as histone modification landscape.…”