Different gene-regulatory elements tend to contain similar or overlapping combinations of transcription factor (TF) binding sites. Thus, it has been difficult to determine how regulatory programmes are encoded. Duttke et al. now show that TFs have position-dependent functions relative to their distance from the transcription start site (TSS). The results suggest how different spatial arrangements of the same TFs might have different regulatory effects.

To investigate how the relative position of TF binding affects function, Duttke et al. knocked down NRF1 and YY1 in U2OS cells and used csRNA-seq to assess changes in transcription initiation. Surprisingly, in both cases, TSSs with increased and decreased activity after knockdown were identified. Notably, TSSs that were downregulated after NRF1 knockdown were enriched for NRF1 binding sites upstream of the TSS, whereas TSSs that were upregulated were enriched for binding sites downstream of the TSS. Thus, although NRF1 is usually considered an activator, its function depends on the position of the respective binding sites. Similarly, for YY1 knockdown, TSSs that were downregulated had binding sites at the preferential location immediately downstream of the TSS, whereas TSSs that were upregulated had binding sites farther from this location. Together, the data show that the position-dependent effects of TFs correlate with the natural enrichment of their binding sites upstream or downstream of the TSS.