Multiplexed dissection of a model human transcription factor binding site architecture

In eukaryotes, transcription factors orchestrate gene expression by binding to TF-Binding Sites (TFBSs) and localizing transcriptional co-regulators and RNA Polymerase II to cis-regulatory elements. The strength and regulation of transcription can be modulated by a variety of factors including TFBS composition, TFBS affinity and number, distance between TFBSs, distance of TFBSs to transcription start sites, and epigenetic modifications. We still lack a basic comprehension of how such variables shaping cis-regulatory architecture culminate in quantitative transcriptional responses. Here we explored how such factors determine the transcriptional activity of a model transcription factor, the c-AMP Response Element (CRE) binding protein. We measured expression driven by 4,602 synthetic regulatory elements in a massively parallel reporter assay (MPRA) exploring the impact of CRE number, affinity, distance to the promoter, and spacing between multiple CREs. We found the number and affinity of CREs within regulatory elements largely determines overall expression, and this relationship is shaped by the proximity of each CRE to the downstream promoter. In addition, while we observed expression periodicity as the CRE distance to the promoter varied, the spacing between multiple CREs altered this periodicity. Finally, we compare library expression between an episomal MPRA and a new, genomically-integrated MPRA in which a single synthetic regulatory element is present per cell at a defined locus. We observe that these largely recapitulate each other although weaker, non-canonical CREs exhibited greater activity in the genomic context.