Interrogating Enhancer Function and Cooperation by Systematic Perturbation of Transposable Elements

项目摘要/摘要 增强子是一种短的非编码DNA元件，它调节基因的表达。他们没有那么多 比启动子和基因保守，它们的出现是新的进化的重要驱动力 表型形式和功能。非编码调控序列的突变会导致许多人类疾病， 包括遗传性孟德尔疾病和癌症。增强剂通常聚集在一起以提供添加剂 远端基因的调控特异性；然而，目前尚不清楚增强子是否与之沟通和合作。 基因组距离以外的其他增强剂。增强剂和促进剂之间的决定性区别是 在启动子的两个不同的转录起始点中，产生信使RNA分子，而 增强剂只产生通常较短且不稳定的非编码转录本。准确地定义了 增强子活性和协作性的语法将有助于理解基础生物学和疾病 人类的病理学。转座元件是可移动的DNA元件，约占人类总数的50% 基因组是物种间和物种内遗传多样性的有效来源，并可通过以下方式发挥增强剂的作用 调控相邻的宿主基因，尽管它们具有诱变潜力。通过描述一群新进化的 具有很高序列相似性的TE衍生增强子，我们将观察到广泛的增强子活性连续体 由每个增强子的基因组背景和生化特性决定。LTR5HS是 最近在人类基因组中内源性的逆转录病毒元件HERV-K，并包含 在人类中是多态的。LTR5HS元件在人类胚胎中转录活跃，并发挥作用 作为控制人类胚胎癌细胞系中275个基因表达的增强剂。我们 将同时和单独地扰乱这些元素，并精确地定义这些元素的影响 对新生转录、3D基因组结构和表型的扰动。首先，我们将使用货物 系统将数十个gRNA导入细胞，结合LTR5HS的序列相似性 Elements允许将dCas9与激活或抑制结构域结合在一起，定位到大多数697 LTR5HS 人类基因组中的元素。在时间轴上，我们将监测新生的转录和长期 元素在扰动后的联系，并推断子网络的活动和协作模式 取决于这些测量的时间和大小。第二，我们将创建池删除 所有LTR5HS元件的文库，并使用单细胞方法测量每个元件对新生转录的影响 从单细胞中捕获gRNA序列和新生RNA并针对gRNA进行选择技术 对生长、分化和多能性表型有害。这种高度细粒度的方法将提供 将增强子的生化特性和基因组背景与转录和转录相关的框架 表型特征。

PROJECT SUMMARY/ABSTRACT Enhancers are short non-coding DNA elements which regulate the expression of genes. They are less conserved than promoters and genes, and their emergence is an important driver of the evolution of new phenotypic forms and functions. Mutations in non-coding regulatory sequences cause many human diseases, including inherited mendelian diseases and cancers. Enhancers often cluster together to confer additive regulatory specificity to a distal gene; however, it is not known if enhancers communicate and cooperate with other enhancers over genomic distance. The defining difference between enhancers and promoters is that one of the two divergent transcription start sites of promoters produces a messenger RNA molecule, while enhancers only produce non-coding transcripts which are often short and unstable. Precisely defining the grammar of enhancer activity and cooperativity will facilitate understanding of the basic biology and disease pathology of humans. Transposable elements are mobile DNA elements which make up ~50% of the human genome, represent a potent source of inter- and intra-species genetic diversity, and can act as enhancers by regulating adjacent host genes despite their mutagenic potential. By characterizing a cohort of newly evolved, TE-derived enhancers with high sequence similarity, we will observe a broad continuum of enhancer activity dictated by the genomic context and biochemical properties of each enhancer. LTR5HS is a subclass of the most recently endogenized retroviral element in the human genome, HERV-K, and contains elements which are polymorphic among humans. LTR5HS elements are transcriptionally active in the human embryo and act as enhancers controlling the expression of 275 genes in a human embryonic carcinoma-derived cell line. We will perturb these elements, both simultaneously and individually, and precisely define the impact of these perturbations on nascent transcription, 3D genome architecture, and phenotype. First, we will use the CARGO system to introduce of tens of gRNAs into cells, which in combination with the sequence similarity of LTR5HS elements enables targeting of dCas9 fused to activating or repressing domains to most of the 697 LTR5HS elements in the human genome. On a temporal axis, we will monitor nascent transcription and long-range contacts of the elements post-perturbation, and infer the mode of activity and cooperation of subnetworks depending on the timing and magnitude of these measurements. Second, we will create a pooled deletion library of all LTR5HS elements and measure the impact of each on nascent transcription using single-cell technology to capture gRNA sequence along with nascent RNA from single cells, and select against gRNAs deleterious for growth, differentiation, and pluripotency phenotypes. This highly granular approach will provide a framework for relating the biochemical properties and genomic context of enhancers to transcriptomic and phenotypic properties.