Systematic discovery of directional regulatory motifs associated with human insulator sites

Insulator proteins act as a barrier of enhancer-promoter interactions (EPIs). CTCF, a DNA-binding protein (DBP) that binds to chromatin interaction anchors, i.e., edges, is the main insulator protein in vertebrates. However, other DBPs involved in insulator function of EPIs are still unclear. Hence, we developed a systematic, comprehensive deep learning-based approach for identifying the DNA motifs of DBPs associated with insulator. 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 in chromatin interaction anchors. Furthermore, we observed that the motifs could regulate transcribed regions differentially repressed in alternative transcripts. These findings reveal that the directional bias of motifs is associated with insulator functions, chromosome conformation and alternative transcription, potentially mediated by structural interactions. These findings contribute to elucidate novel transcriptional regulations. ### Competing Interest Statement The authors have declared no competing interest.