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特定的基因模式 通过编码转录因子的结合位点来表达。然而，大型基因组包含 转录因子的短的、简并的序列基序的数百万个虚假副本 认识到。人们还不知道真正的功能结合位点是如何与 非功能性的图案。这是一个关键的悬而未决的问题，因为越来越多的证据表明 破坏或创建转录因子结合位点的遗传变异在 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异体。 为了解决这个问题，我们的长期目标是了解活性转录因子是如何结合的 位点由它们的DNA序列特征来确定。最近的结果表明，功能性的 结合位点与虚假基序的区别在于关键的局部序列位于 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 在这个广泛的问题上，我们将研究光感受器转录因子CRX的结合位点。 我们的具体目标是：第一，了解侧翼dna序列特征如何区分 来自含有虚假CRX的非活性基因组序列的转录活性CRX结合位点 图案。其次，为了量化和模拟局部侧翼序列特征的影响，使用 人工合成CRX结合位点的易处理系统。这项建议的主要创新之处在于 测量大量野生型转录因子结合和顺式调节活性， 突变和合成序列，从而直接量化侧翼之间的关系 DNA序列、结合和活性。使用最近开发的高通量分析来 测量顺式调节活性和CRX结合亲和力，我们将直接测试其功能作用 通过破坏CRX结合位点的侧翼序列特征来获得局部侧翼序列。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序列特征是如何组合在一起来指定活性的 基因组中的CRX位点。这一结果将提高我们对序列变异如何 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.