Ceolin, S. | Search bioRxiv

bioRxiv2024

Entangled and non-modular enhancer sequences producing independent spatial activities

The modularity of transcriptional enhancers (1, 2) is central to our understanding of morphological evolution, as it allows specific changes to a gene expression pattern component, without affecting others (3). Enhancer modularity refers to physically separated stretches of regulatory sequence producing discrete spatiotemporal transcriptional activity. This concept stems mainly from assays that test the sufficiency of a DNA segment (4) to drive spatial reporter expression resembling that of the corresponding gene. Focusing on sufficiency to produce spatial patterns, it overlooks quantitative aspects of gene expression, underestimating the regulatory sequence actually required to reach full endogenous expression levels. Here we show that five regulatory activities of the pigmentation gene yellow in Drosophila, classically described as modular (5-7), result from extensively overlapping sequences, with broadly distributed regulatory information. Nevertheless, the independent regulatory activities of these entangled enhancers appear to be nucleated by specific segments that we called enhancer cores. Our work calls for a reappraisal of enhancer definition and properties, as well as of the consequences on regulatory evolution.

developmental biology↗

bioRxiv2020

Context dependent activation/repression by Hunchback binding sites in Drosophila embryo

Hunchback (Hb) is considered a context-dependent transcription factor, able to activate or repress different enhancers during Drosophila embryo segmentation. The mechanism driving the contextdependent activity of Hb is however not well understood. Here we measure the activity of a large set of 20 synthetic enhancers that we design to elucidate the effect of Hb binding sites in Drosophila segmentation. We obtain quantitative data on the spatiotemporal dynamics of activity of all synthetic enhancers in-vivo, by using a quantitative and sensitive reporter system we recently developed. Our data reveal the dual role of Hb binding sites in segmentation enhancers: on the one hand, Hb act as a typical short range repressor by binding to its cognate sequences; on the other hand, we report a novel effect of a sequence containing multiple Hb binding sites, which is able to increase enhancer activity independently from Hb binding. This sequence, which contains multiple Poly-dA stretches, increases the activity of enhancers driven by different activators, possibly by disfavoring nucleosome occupancy. AUTHOR SUMMARYThe control of gene expression is a fundamental process that allows cells to respond to external stimuli and take on various identities in complex organisms. Enhancers are DNA sequences that play a key role in this process. In the simplest model of an enhancer, small parts of its sequence can be specifically bound by proteins called transcription factors and the occupancy pattern of these proteins on the enhancer determines the expression level of a specific gene. In this research work we have studied enhancers in the context of the development of a fruit fly embryo. We have built synthetic enhancer sequences containing binding sites for a few specific factors and measured their activity in living embryos using fluorescence microscopy. Our results revealed that binding sites for a particular protein, Hunchback, are able to influence the activity of the enhancer even independently from Hunchback binding to them. This discovery might help to explain the complex effects that have been observed when studying Hunchback binding sites in natural enhancers.

developmental biology↗