Webinar by Arnaud Krebs
Cooperativity and antagonism at cis-regulatory elements
Genome Biology, EMBL, Heidelberg, Germany
Arnaud Krebs: Cis-regulatory elements (CREs) control the expression of genes involved in the acquisition of cellular identity during development, and its maintenance in healthy tissues. CRE activation typically requires the binding of multiple TFs, yet the precise mechanisms underlying their coordinated action are largely unknown. TFs bind their target motifs in the context of chromatin, that carries information regulating their action. The impact of these epigenetic signals on the function of TFs at CREs is largely unknown. Current bulk assays used to map TF occupancy average binding events arising from millions of individual cells, not informing on the potential cooperativity and the antagonisms that organize their binding at CRE. To move beyond this boundary, we developed Single Molecule Footprinting (SMF) to quantify the binding of TFs at mouse regulatory regions. The method allows to simultaneously measure the occurrence of multiple TFs, nucleosomes and DNA methylation on individual molecules genome wide. I will illustrate how we leveraged this new layer of information to understand mechanisms of TF cooperativity and the epigenetic regulation of TF binding at enhancers. Detecting multiple TF binding events on single DNA molecules has enabled us to determine TF co-binding frequencies in vivo. Systematic analysis of the occupancy patterns of thousands of TF pairs reveals widespread evidence of cooperative binding. It elucidates the binding cooperativity mechanism used by transcription factors in absence of strict organisation of their binding motifs, a characteristic feature of most of enhancers. Simultaneously quantification of DNA methylation and TF binding on individual DNA molecules allowed us to test if TF binding can occur at enhancers when their target DNA molecules are methylated in vivo. We identified a subset of cell type specific enhancers that showed reduced accessibility on methylated molecules, indicating a possible regulation of these loci by DNA methylation. Genetic perturbation experiments suggest a direct epigenetic control of TF binding at these loci.