SRA

Enhancers and silencers often depend on the same transcription factors (TFs) and are imperfectly distinguished from each other by genomic assays of TF binding or chromatin state. To identify sequence features that define enhancers and silencers, we assayed massively parallel reporter libraries of genomic sequences targeted by the photoreceptor TF CRX and found instances of enhancer, silencer, or no activity. Both enhancers and silencers contain more TF motifs than inactive sequences, but enhancers contain motifs from a more diverse collection of TFs. We developed a measure of information content that describes the number and diversity of motifs in a sequence and found that, while both enhancers and silencers depend on CRX motifs, enhancers have higher information content. Our results indicate that enhancers contain motifs for a diverse but degenerate collection of TFs, while silencers depend on a smaller and less diverse collection of TFs. Overall design: Massively Parallel Reporter Assays of CRX-targeted cis-regulatory sequences in mouse retinas. Each unique sequence is tagged with 3 unique 9 bp barcodes. Each library also includes a basal control (basal promoter only) tagged with 18 unique 9 bp barcodes. Libraries contain genomic sequences centered on CRX motifs and versions of the same sequences with all CRX motifs abolished by point mutation. Libraries were cloned upstream of the Rho promoter or a minimal Polylinker. Libraries were electroporated into retinas dissected from P0 CD-1 mice. Retinas were harvested at P8. Barcode cDNA and the original DNA plasmids were sequenced. Three replicates per experiment.