Discovery of biased orientations of regulatory motifs affecting transcription of human genes and including known insulators

Chromatin interactions play essential roles in enhancer-promoter interactions (EPIs) and regulating the transcription of genes. CTCF and cohesin proteins are located at the anchors of chromatin interactions, forming their loop structures. Other DNA binding proteins such as YY1, ZNF143 and SMARCA4 are also reported to be involved in chromatin interactions. However, the overall picture of proteins associated with chromatin interactions and insulator function is still unclear. Here I describe a systematic and comprehensive approach to discover DNA binding motif sequences of transcription factors (TFs), affecting the interaction between enhancers and promoters of genes and their expression by the insulator function of the TFs. This analysis identified 96 biased orientations of DNA motifs of TFs that affected the expression level of putative transcriptional target genes significantly in monocytes, T cells, HMEC and NPC in common, and included known TFs involved in chromatin interaction and insulator function such as CTCF, cohesin (RAD21 and SMC3), YY1 and ZNF143. The 96 biased orientations of DNA motifs contain 64 forward-reverse (FR) and 52 reverse-forward (RF) orientations of DNA motifs (Some TFs have more than one DNA motifs in databases, so the total number is smaller than the sum of FR and RF). To confirm the predicted DNA motifs, first, I compared with chromatin interaction data. Despite the limitation of resolution of chromatin interaction data, for 44 (69%) FR and 28 (54%) RF orientations of DNA motif sequences in CD4+ T cells, EPIs predicted based on an enhancer-promoter association (EPA) that was shortened at the biased orientations of DNA motif sites, overlapped with chromatin interactions significantly more than other types of EPAs. Second, using gene expression data among 53 tissues, 43 (72%) FR and 40 (80%) RF orientations of DNA motifs showed significantly reduced correlation in expression level of nearby genes separated by the motif sites. Some DNA motif sites of biased orientations of DNA motifs of TFs were found between nearby genes not including DNA motif sites of the known TFs involved in chromatin interactions and between low correlation of expression level of nearby genes. These analyses suggest that the DNA motifs are associated with insulator functions.