Transcription factor binding site orientation and order are major drivers of gene regulatory activity

The gene regulatory code and grammar remain largely unknown, precluding our ability to link phenotype to genotype in regulatory sequences. Here, using a massively parallel reporter assay of 209,440 sequences, we examine all possible pair and triplet combinations, permutations and orientations of eighteen liver-associated transcription factor binding sites (TFBS). We find that TFBS orientation and order have a major effect on gene regulatory activity. Corroborating these results with genomic analyses, we find clear promoter TFBS orientation biases which significantly influence expression levels of the downstream genes they regulate. Additionally, taking into account TFBS orientation in a model that predicts expression from sequence improves performance by 19%. Collectively, our results show that TFBS orientation and order have a significant effect on gene regulatory activity and should be taken into account when analyzing the functional effect of variants on the activity of these sequences.