PRE2023-In vivo determinants of 3D enhancer-promoter communication-PID2022-143253NB-I00

During development, a temporal and spatial control of gene expression is key for the appropriate differentiation of cellular types, tissues and organs. In animals, transcription is largely controlled by cis-regulatory elements, such as enhancers. These elements and their cognate gene promoters are generally framed within topologically associating domains (TADs) spatial chromatin domains that facilitate the functional interaction between these elements. However, additional mechanisms can also contribute to enhancer-promoter specificity and those are not well understood. In this proposal, we aim to dissect the regulatory interplay between promoters and the architectural factor CTCF in ensuring correct gene expression during development. For that, we will focus on studying the regulation of the Epha4 locus in the context of mouse limb development. This experimental design has the advantage that it can measure the effects of genomic perturbations in vivo at three different level: 3D chromatin interactions, gene expression and phenotypes. By generating and analyzing a series of 11 mutant mouse backgrounds, we will (1) dissect the relative contribution of CTCF binding sites (CBS) and promoters in insulator function; (2) Investigate how CBS and promoter functionality modulates selective gene activation; and (3) Determine how changes in canonical transcription relate to non-target gene activation.