Systematic dissection of Transcription Factor binding cooperativity mechanisms

• Applicant: Arnaud Krebs, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 446603164

Precise execution of transcriptional programs is required for faithful acquisition of cell identity during development. Gene expression patterns defining this identity are largely regulated by transcription factors (TFs). TFs bind to sequence motifs contained in cis-regulatory elements (CREs), leading to the recruitment of cofactors that directly modulate the activity of target genes. Activation of a CRE typically requires the binding of multiple TFs with various functions including remodelling of their chromatin and specification of their activity. Therefore, cooperativity between TFs is assumed to be essential for transcription regulation. Multiple complementary mechanisms have been proposed to explain this phenomenon, however their precise role in controlling CREs activity remains largely unknown. Understanding how TF cooperativity contributes to gene expression requires an experimental system to selectively perturb binding of individual TFs and quantify the consequences on collective TF binding as well as the transcriptional activity of CREs. Current approaches used to map TF binding are based on the selective enrichment of the DNA bound by individual TFs, not providing information on the degree of co-binding between TFs. Here, we propose to move beyond this boundary using Single Molecule Footprinting (SMF), a method that we recently demonstrated to be able to simultaneously quantify the occupancy by multiple TFs at CREs. We will leverage natural genetic variation existing between distant mouse species to study the effect of discrete changes in the affinity of TF binding sites across the genome. We will quantify collective TF binding patterns using SMF in four different genotypes that diverge by a high number of single nucleotide variants. The generated data will allow us to analyse the effects of the genetic alterations of thousands of individual binding sites on collective TF binding at CREs. We will study the consequences of these changes on the activity of CREs in mouse embryonic stem cells and during their commitment to neurons. This genome-scale perturbation experiment will enable us to globally identify the mechanisms underlying TF cooperativity at CREs and model their contribution to cell fate choices. Importantly, we will further test predictions of our model by measuring TF binding and transcriptional activity of thousands of synthetic CRE variants carrying designed mutations. Together, the proposed work will characterise how TF cooperativity mechanisms contribute to transcriptional control of cellular identity during differentiation.

• DFG Programme: Research Grants