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 99 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. Furthermore, we observed that the motifs could regulate transcribed regions differentially repressed in alternative transcripts. These analyses reveal that the directional bias of motifs is associated with insulators, chromosome conformation and alternative transcription, potentially mediated by structural interactions. These findings contribute to elucidate novel transcriptional regulations.