Quantification of mouse retinal enhancer activity by massively parallel reporter assay

Cone-rod homeobox (CRX) is a paired-like homeodomain transcription factor (TF) and a master regulator of photoreceptor development in vertebrates. The in vitro DNA binding preferences of CRX have been described in detail, but the degree to which in vitro binding affinity is correlated with in vivo enhancer activity is not known. In addition, paired-class homeodomain TFs can bind DNA cooperatively as both homodimers and heterodimers at inverted TAAT half-sites separated by two or three nucleotides. This dimeric configuration is thought to mediate target specificity, but whether monomeric and dimeric sites encode distinct levels of activity is not known. Here, we use a massively parallel reporter assay, CRE-seq, to determine how local sequence context shapes the regulatory activity of CRX binding sites in mouse photoreceptors. We assay inactivating mutations in >1700 TFBSs, and we find that dimeric CRX binding sites act as stronger enhancers than monomeric CRX binding sites. Furthermore, the activity of dimeric half-sites is cooperative, dependent on a strict three-base-pair spacing, and tuned by the identity of the spacer nucleotides. Saturating single-nucleotide mutagenesis of 195 CRX binding sites shows that, on average, changes in TFBS affinity are correlated with changes in regulatory activity, but this relationship is obscured when considering mutations across multiple CREs. Taken together, these results demonstrate that the activity of CRX binding sites is highly dependent on sequence context, providing insight into photoreceptor gene regulation and illustrating functional principles of homeodomain binding sites that may be conserved in other cell types. CRE-seq of mouse retinal explants. 100,000 100-bp target seqeunces were cloned upstream of a photoreceptor promoter (either pRho or pCrx) driving a DsRed reporter harboring CRE-specific 13-bp barcodes in the 3' UTR. This library was electroporated into intact retinas from newborn (P0) mice. Retinas were grown in culture for eight days, at which point RNA and DNA were harvested and barcodes were amplifieid and sequenced to quantify enhancer activity. Each biological replicate consisted of five electroporated retinas, and three biological replicates were performed for each condition (assaying constructs on either pRho or pCrx).