Project 4 Functional Dissection of Erythroid Super-Enhancer, Daniel E. Bauer

摘要 增强子是位于正向调节基因表达的基因附近的非编码DNA元件。 因为增强剂通常以特定细胞类型的方式起作用，它们赋予基因生物学特异性。 监管。增强子中常见的遗传变异似乎是性状关联的一种普遍手段。 超级增强剂是最近描述的一组高度富含细胞调节元件的增强剂 与谱系特定疾病易感性相关的谱系定义基因和遗传变异。然而， 超强启动子功能的结构决定因素仍然知之甚少。增强器通常定义为 通过它们相关的生化特征或报告分析中的异位潜势。尽管如此，黄金标准 增强子活性测试是功能缺失突变，以识别对调控序列的要求 它们的自然染色体环境。我们的团队已经开发出一种由Cas9介导的原位饱和 突变技术，允许高分辨率、高通量的增强子功能分析 原生基因组环境。该分析涉及设计和合成饱和感兴趣区域的引导RNA 随后进行慢病毒CRISPR联合筛查。这种方法与基因组靶深度结合时 测序允许对关键的功能序列进行接近核苷酸分辨的评估 增强功能。在这项建议中，我们应用这项技术系统地扰乱了20个红系特异性 关键红系基因的超级增强子。我们通过使用一个变种来扩展Cas9突变策略 Cas9，以提高基因组编辑分辨率。此外，我们还采用了变量信息导引设计 即使面对自然的基因组变异也要改进诱变的方法。我们利用生物信息学 识别基本序列，预测相互作用的转录因子，并改进模型进行估计的方法 增强剂活性。我们使用生化技术来确定转录因子和 染色质调节剂在所需的序列。我们专注于定义和测试超级增强器循环 关键转录因子GATA1和TAL1参与的相互作用。最后，我们聘请了潜在客户 反向遗传学验证超级增强子所需的顺式作用序列和反式作用因子 功能。这些研究将有助于确定集群组件在 超级增强剂。我们希望这些实验能让我们进一步了解 红细胞生成以及基因调控的基本机制。

ABSTRACT Enhancers are non-coding DNA elements near genes that positively regulate gene expression. Because enhancers often act in a cell type specific manner, they confer biological specificity on gene regulation. Common genetic variation within enhancers appears to be a prevalent means of trait association. Super-enhancers are a recently described set of enhancers highly enriched for regulatory elements of cell lineage-defining genes and genetic variants related to lineage-specific disease susceptibility. However the structural determinants of super-enhancer function remain poorly understood. Enhancers are typically defined by their correlated biochemical features or ectopic potential in reporter assays. Nonetheless the gold-standard test of enhancer activity is loss-of-function mutation to discern the requirement for regulatory sequences in their natural chromosomal environment. Our group has developed a Cas9-mediated in situ saturating mutagenesis technique that allows for high-resolution, high-throughput functional analysis of enhancers in the native genomic setting. The assay involves design and synthesis of guide RNAs saturating a region of interest followed by pooled lentiviral CRISPR screening. This method when coupled with genomic target deep sequencing allows for an evaluation nearing nucleotide resolution of the functional sequences critical for enhancer function. In this proposal, we apply this technique to systematically perturb twenty erythroid-specific super-enhancers of key erythroid genes. We extend the Cas9 mutagenesis strategy by making use of a variant Cas9 to allow for increased genome editing resolution. In addition, we take a variant-informed guide design approach to improve mutagenesis even in the face of natural genomic variation. We utilize bioinformatic methods to identify essential sequences, predict interacting transcription factors, and refine models to estimate enhancer activity. We use biochemical techniques to determine the occupancy of transcription factors and chromatin regulators at required sequences. We focus on defining and testing super-enhancer looping interactions in which the key transcription factors GATA1 and TAL1 participate. Finally we employ prospective reverse genetics to validate both cis-acting sequences and trans-acting factors necessary for super-enhancer function. These studies will help determine emergent cooperative properties of clustered components within super-enhancers. We expect these experiments to inform a further understanding of crucial aspects of erythropoiesis as well as fundamental mechanisms of gene regulation.