Information Content Differentiates Enhancers From Silencers in Mouse Photoreceptors

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. 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.

增强子与沉默子通常依赖于同一套转录因子（Transcription Factors, TFs），且通过转录因子结合或染色质状态的基因组检测手段无法对二者进行完美区分。为鉴定定义增强子与沉默子的序列特征，我们针对光感受器转录因子CRX靶向的基因组序列构建了大规模平行报告基因文库并开展检测，发现了具有增强子活性、沉默子活性或无活性的序列片段。相较于无活性序列，增强子与沉默子均含有更多的转录因子基序（TF motifs），但增强子所含基序来自更为多样的转录因子集合。我们开发了一种信息含量测量方法，用于描述序列中基序的数量与多样性，研究发现尽管增强子与沉默子均依赖CRX基序，但增强子的信息含量更高。本研究结果表明，增强子含有来自多样但简并的转录因子集合的基序，而沉默子则依赖于规模更小、多样性更低的转录因子集合。本数据集对应小鼠视网膜中CRX靶向顺式调控序列的大规模平行报告基因检测实验：每条独特的基因组序列均带有3个唯一的9bp条形码标签；每个文库还包含仅含基础启动子的阴性对照，该对照带有18个唯一的9bp条形码标签。文库包含以CRX基序为中心的基因组序列，以及通过点突变完全消除所有CRX基序的对应序列变体。文库被克隆至Rho启动子或最小化多克隆位点（Polylinker）的上游区域。将文库通过电穿孔法转入从P0龄CD-1小鼠体内分离的视网膜组织中，在P8龄时收集视网膜组织。对条形码cDNA以及原始DNA质粒进行测序，每个实验设置3次生物学重复。