Systematic discovery of directional regulatory motifs associated with human insulator sites

Insulator proteins act as a barrier of enhancer-promoter interactions (EPIs). The main insulator protein in vertebrates is CTCF, a DNA-binding protein (DBP). However, other DBPs associated with insulators of EPIs are still unclear. Hence, we developed a systematic, comprehensive deep learning-based approach for identifying the DNA motifs of DBPs associated with insulators. We discovered 97 directional and minor nondirectional motifs in human fibroblast cells that corresponded to 23 DBPs related to insulator function, CTCF, and/or other types of chromosomal transcriptional regulation reported in previous studies. The estimated CTCF orientation bias was consistently proportional to CTCF orientation bias observed in chromatin interactions. The motifs were significantly more abundant at insulator sites separated by repressive and active regions, at boundary sites identified by chromatin interaction data, and at splice sites than other DBPs. For instance, we found that the key regulator MyoD-binding site is located at an insulator site near a gene involved in skeletal muscle differentiation and function. Finally, the insulator-pairing model explains that homologous and heterologous insulator-insulator pairing interactions are orientation-dependent. These findings contribute to elucidate novel transcriptional regulations.