Mammalian genomes are divided into Topologically Associated Domains (TADs) that restrict the formation of Enhancer-Promoter loops and other biological processes. TADs are formed by a process of Cohesin-mediated loop extrusion, which is subsequently blocked at defined TAD boundaries. Most TAD boundaries bind the CTCF insulator protein, but features like G-quadruplexes (G4s) and transcriptional start sites are enriched as well. We recently reported that CTCF binding is clustered at most TAD boundaries, both at the level of ChIP-seq peaks and of DNA binding motifs within peaks. Clustering of binding motifs within peaks was particularly beneficial for CTCF binding at lower affinity motifs, suggesting that this local clustering may create binding synergies. Using Nano-C, a new multi-contact 3C assay, we showed that TAD boundaries are not impermeable and that CTCF binding peaks contributed individually (but incompletely) to the blocking of loop extrusion. Clustering of peaks thereby improved the overall capacity for loop extrusion blocking, thereby enhancing the separation between TADs. We hypothesize that loop extrusion blocking at TAD boundaries can be defined by their grammar of CTCF binding (‘Insulator Grammar'), with chromatin features like nucleosome positioning, transcription and G4s having a further impact. This DNA-encoded grammar provides a flexible means for the regulation of TAD boundary permeability, thereby allowing the fine-tuning of enhancer-promoter loop formation and gene regulation. Our InsulatorGrammar project aims to systematically dissect how the CTCF grammar can be used to regulate Cohesin-mediated loop extrusion, thereby allowing the modulation of enhancer-promoter loop formation. To address this aim, we will determine how the DNA-encoded local clustering of CTCF binding motifs, in combination with other chromatin features, creates favorable chromatin environments for CTCF binding, and how this synergy affects loop extrusion and enhancer blocking. First, we will first use cells that permit a timed degradation and reestablishment of the CTCF protein to determine, genome-wide, how the CTCF binding grammar (number, orientation and affinity of motifs) and other chromatin features reciprocally influence CTCF binding. Next, we will use a newly developed reporter system for enhancer-promoter looping to integrate 50 synthetic ‘designer’ insulator elements that vary for the number, orientation and affinity of CTCF motifs, and in the presence or absence of nearby transcription and G4s. For all lines, the influence of the CTCF grammar on CTCF binding and enhancer blocking will be determined. Moreover, one-third of these lines will be analyzed using a strategy that combines in-depth multi-omics with biophysical modeling to unravel underlying characteristics of chromatin organization. These studies will reveal how the CTCF grammar and the surrounding chromatin features reciprocally affect the DNA binding of CTCF, the positioning of nucleosomes and the formation of G4s. These structural insights will subsequently be linked to the functional impact on loop extrusion blocking, enhancer-promoter looping and TAD organization. Our multi-disciplinary InsulatorGrammar project will generate an unprecedented view of how clustering of CTCF binding sites can synergize with other chromatin features to create a DNA-encoded means for the fine-tuning of enhancer-promoter looping and gene regulation. The results will help to explain how more subtle changes in gene activity can be introduced, with relevance for development, evolution and disease.