DISCOVERY OF DNA DETERMINANTS OF TRANSCRIPTION FACTOR BINDING AND FUNCTION IN PHO

项目摘要 非蛋白质编码基因组的一个主要功能是指导基因的特定模式， 通过编码转录因子的结合位点来表达。然而，大基因组包含 数以百万计的虚假拷贝的短，简并序列基序，转录因子 认识。目前还不清楚如何区分真正的功能性结合位点与 非功能性图案。这是一个尚未解决的关键问题，因为越来越多的证据表明， 破坏或产生转录因子结合位点的遗传变异在基因组中广泛存在， 在疾病中的作用，但我们目前不能准确地识别影响活性结合位点的变体。 为了解决这个问题，我们的长期目标是了解激活的转录因子如何结合 位点由它们的DNA序列特征指定。最近的研究结果表明， 结合位点与假基序的区别在于侧翼的关键局部序列， 核心主题使用哺乳动物视网膜作为生理相关的模型系统来解决 这个广泛的问题，我们将研究感光转录因子CRX的结合位点。 我们的具体目标是：首先，了解侧翼DNA序列特征如何区分 来自具有假CRX的非活性基因组序列的转录活性CRX结合位点 图案第二，为了量化和模拟局部侧翼序列特征的影响， 合成CRX结合位点的易处理系统。该提案的主要创新之处在于， 在大量野生型上测量转录因子结合和顺式调节活性， 突变体和合成序列，从而直接量化侧翼序列之间的关系。 DNA序列、结合和活性。使用最近开发的高通量检测， 测量顺式调节活性和CRX结合亲和力，我们将直接测试功能作用 通过破坏CRX结合位点的侧翼序列特征来改变局部侧翼序列。通过 结合功能基因组学和合成生物学研究天然和合成CRX 结合位点，我们将发现不同的DNA序列特征如何联合收割机组合以指定活性 基因组中的CRX位点。这一结果将提高我们对序列变异 外部核心基序影响转录因子结合位点。

Project Summary A major function of the non-protein-coding genome is to direct specific patterns of gene expression by encoding binding sites for transcription factors. However, large genomes contain millions of spurious copies of the short, degenerate sequence motifs that transcription factors recognize. It is not understood how genuinely functional binding sites are distinguished from non-functional motifs. This is a critical unsolved problem, because growing evidence indicates that genetic variants that disrupt or create transcription factor binding sites play a widespread role in disease, but we currently cannot accurately identify variants affect active binding sites. To address this issue, our long term goal is to understand how active transcription factor binding sites are specified by their DNA sequence features. Recent results suggest that functional binding sites are distinguished from spurious motifs by critical local sequences that flank the core motif. Using the mammalian retina as a physiologically relevant model system to address this broad issue, we will investigate binding sites for the photoreceptor transcription factor CRX. Our specific aims are: First, to understand how flanking DNA sequence features distinguish transcriptionally active CRX binding sites from inactive genomic sequences with spurious CRX motifs. Second, to quantify and model the effects of local flanking sequence features using a tractable system of synthetic CRX binding sites. The major innovation of this proposal is to measure both transcription factor binding and cis-regulatory activity on a large set of wild-type, mutant, and synthetic sequences, and thereby directly quantify the relationship between flanking DNA sequence, binding, and activity. Using recently developed high-throughput assays to measure cis-regulatory activity and CRX binding affinity, we will directly test the functional role of local flanking sequences by disrupting flanking sequence features of CRX binding sites. By combining functional genomics and synthetic biology to investigate natural and synthetic CRX binding sites, we will discover how different DNA sequence features combine to specify active CRX sites in the genome. The result will improve our understanding of how sequence variants outside core motifs affect transcription factor binding sites.