Molecular mechanisms of enhancer-promoter communication during development

• Applicant: Arnaud Krebs, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 522185250

Binding of Transcription Factors (TFs) at enhancers is a key step for the activation of the genes required for the acquisition of cell identity during development. Enhancers are often located several mega-bases away from the gene they regulate. Thus, activation of most developmental genes requires their long-range interactions with their target promoter. Yet, the mechanisms by which the binding of TFs at enhancers contribute to the formation of enhancer-promoter contacts, and the later activation of transcription are poorly understood. Is the binding of TFs at enhancers stimulating the recruitment of TFs at promoters? Is this coordinated recruitment required for the formation of E-P contacts? Is coordinated TF binding at E-P pairs required for the recruitment of RNA Pol II and the activation of transcription? To address these questions, we will develop a technology that will detect the genome occupancy of TFs at enhancers and promoters simultaneously. We will combine DNA footprinting of TFs using methyl-transferases with long DNA molecule sequencing, to generate continuous DNA occupancy maps over 15 kb. We will perform this experiment in two paradigm of gene activation 1) hormone induction of gene activation 2) at two developmental stages of Drosophila larvae, tow well-established model for the study of gene regulation mechanisms. In Drosophila, a majority of enhancers are located within 7 kilobases of their target promoter. This will allow us to ask if the binding of certain TFs at enhancers, lead to changes in TF binding and transcriptional activation at promoters. Our data will identify coordinated TF binding between distant regulatory elements, that may indicate their involvement in the communication of activation signals at promoters. Many of the genes that are expressed at late stages of development, already form E-P contacts hours before activation of transcription at their promoters. We will use this temporal disconnection to ask if the TF co-occupancy observed between distant sites is more likely to be involved in E-P formation or transcription activation. To move beyond correlative evidences, we will perturb TF binding at these sites in cis and trans. We will combine the use of rapid depletion of the TFs with optogenetics and site-specific mutagenesis using CRISPR-Cas9, and probe the consequences on the formation of E-P contacts and transcription acti cis und trans stören. Wir werden den schnellen Abbau der TFs mit Optogenevation. This data will establish the involvement of specific TFs play a role in the formation of E-P contacts. Moreover, it will identify if the simultaneous binding of certain combinations of TFs at E-P pairs activates transcription. Together, this study will identify the fundamental mechanisms allowing enhancers to communicate their activation signal at promoters during embryonic development.

• DFG Programme: Research Grants