Position-dependent function of human sequence-specific transcription factors

Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, often contain similar assortments of sequence-specific transcription factor (TF) binding sites. Knowledge of how these sequence motifs encode multiple, often overlapping gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease. Exploiting natural genetic variation, perturbation of endogenous TF protein levels, and analysis of synthetic regulatory elements using a novel transcription start site (TSS) capturing massively parallel reporter assay approach, here we show that the outcome of TF binding on transcription initiation is position dependent. Analyzing TF motif occurrences relative to the TSS, we identified several motifs with highly preferential and superhelical positioning. We show that these patterns are a combination of a TF's distinct functional profiles: many TFs, including canonical activators like NRF1, NFY, Sp1 or YY1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs can collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TFs, but with different spatial relationships, can result in distinct gene regulatory outcomes and underscore a critical role for TSS data in decoding the regulatory information of our genome.