Deciphering the regulatory logic of aDrosophilaenhancer through systematic sequence mutagenesis and quantitative image analysis

Abstract

Transcriptional enhancers are short DNA sequences controlling the spatial activity, timing and levels of eukaryotic gene transcription. Their quantitative transcriptional output is thought to result from the number and organization of transcription factor binding sites (TFBSs). Yet, how the various aspects of regulatory information are encoded in enhancer sequences remains elusive. We addressed this question by quantifying the spatial activity of the yellow spot enhancer active in developing Drosophila wings. To identify which enhancer DNA sequence contributes to enhancer activity, we introduced systematic mutations along the enhancer. We developed an analytic framework that uses comprehensive descriptors to quantify reporter assay in transgenic Deciphering the regulatory logic of an enhancer 2 flies and measure spatial variations in activity levels across the wing. Our analysis highlights an unexpected density of regulatory information in the spot enhancer sequence. Furthermore, it reveals an unanticipated regulatory logic underlying the activity of this enhancer, and how it reads the wing trans-regulatory landscape to encode a spatial pattern.Deciphering the regulatory logic of an enhancer

Ancestral and derived transcriptional enhancers share regulatory sequence and a pleiotropic site affecting chromatin accessibility

“…As the effect on the wing blade activity is not observed in D2 block4 , which also randomizes site spot 196 [6] , it is likely that sites for repressors acting on both activities are located within the 100 bp randomized in D2 block4 . In our separate dissection of spot 196 ( 28 ), we reached a similar conclusion for the role of spot 196 [6] . Even without knowing the molecular mechanism at work, our results suggest that spot 196 [6] could be the target site of a global, permissive activator of both activities in the context of segment spot 196 .…”

“…3 ). In a recent and independent dissection of spot 196 , we identified a potential site for one or more unknown transcription factor(s), spot 196 [6] , whose mutation (12 bp) nearly abolishes spot 196 activity completely ( 28 ). It is conceivable that these sites necessary for the spot activity also influence the wing blade activity, thereby producing pleiotropic effects.…”

“…( D ) Mutating all four characterized Dll binding sites ( 16 ) of spot 196 in the context of D2 ( D2 Dll-KO ) reduces the spot activity strongly and the wing blade activity moderately, as seen when comparing this mutant construct with D5 . ( E ) Mutating a newly identified activator site ( 28 ) of spot 196 ( spot 196 [6] , 12 bp mutated) in the context of D2 ( D2 [6]-KO ) reduces both spot and wing blade activities strongly, as seen when comparing this mutant construct with D5 . ( F ) Chromatin accessibility measured with ATAC-seq at the D2 and D2 [6]-KO transgenes at the onset of spot activity ( SI Appendix , Fig.…”

Mutational scans reveal differential evolvability ofDrosophilapromoters and enhancers

“…2A ). Together, these results are consistent with the previous finding that enhancers encode dense spatial information (Fuqua et al 2020; Le Poul et al 2020; Galupa et al 2023).…”

“…We found that all ten lines had reduced number of nuclei expressing lacZ [ Fig. 2A-C, Fig S2 , also see (Fuqua et al 2020; Galupa et al 2022) for complete analysis of the E3N library], consistent with the previous finding that enhancers encode dense spatial information (Fuqua et al 2020; Le Poul et al 2020; Galupa et al 2022). Seven of them also showed reduced level of expression (FDR-adjusted p < 0.05, Wilcoxon test, Fig.…”