Transcription factor interactions explain the context-dependent activity of CRX binding sites

The effects of transcription factor binding sites (TFBSs) on the activity of a cis-regulatory element (CRE) depend on the local sequence context. In rod photoreceptors, binding sites for the transcription factor (TF) Cone-rod homeobox (CRX) occur in both enhancers and silencers, but the sequence context that determines whether CRX binding sites contribute to activation or repression of transcription is not understood. To investigate the context-dependent activity of CRX sites, we fit neural network-based models to the activities of synthetic CREs composed of photoreceptor TFBSs. The models revealed that CRX binding sites consistently make positive, independent contributions to CRE activity, while negative homotypic interactions between sites cause CREs composed of multiple CRX sites to function as silencers. The effects of negative homotypic interactions can be overcome by the presence of other TFBSs that either interact cooperatively with CRX sites or make independent positive contributions to activity. The context-dependent activity of CRX sites is thus determined by the balance between positive heterotypic interactions, independent contributions of TFBSs, and negative homotypic interactions. Our findings explain observed patterns of activity among genomic CRX-bound enhancers and silencers, and suggest that enhancers may require diverse TFBSs to overcome negative homotypic interactions between TFBSs. Transcription factors control gene expression in different cell types by binding to sites in regulatory DNA. The same transcription factor when bound at different DNA sites will have different effects on gene expression, but how a single factor can produce divergent effects is unclear. The photoreceptor transcription factor CRX activates expression from regulatory DNA that harbors few copies of a CRX binding site, while it represses expression when many binding site copies are present. We modeled how the number and arrangement of binding sites for CRX and other factors affect gene expression, using data from libraries of synthetic regulatory DNA elements. The model shows that individual transcription factor binding sites increase expression on their own, but interactions between multiple copies of the same site decrease expression. Our results generalize across transcription factors and tissues, suggesting that this is a general principle that might help explain differing patterns of expression across tissues. The model explains how interactions between binding sites allow a single transcription factor to have contrasting effects on gene expression in the same cell type.