Interrogating Enhancer Function and Cooperation by Systematic Perturbation of Transposable Elements

项目总结/摘要 增强子是调节基因表达的短的非编码DNA元件。他们不太 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导致许多人类疾病， 包括遗传性孟德尔疾病和癌症。增强子通常聚集在一起， 调控特异性的远端基因;然而，它是不知道，如果增强子通信和合作， 其他基因组距离的增强子。增强子和启动子之间的决定性区别在于 启动子的两个不同的转录起始位点产生信使RNA分子， 增强子仅产生非编码转录物，其通常短且不稳定。精确定义 增强子活性和协同性的语法将有助于理解基础生物学和疾病 人类的病理学转座因子是一种移动的DNA因子，占人类基因组的50 基因组，代表了物种间和物种内遗传多样性的一个潜在来源，并可以作为增强子， 调节邻近宿主基因，尽管它们具有致突变潜力。通过描述一群新进化的， TE衍生的增强子具有高序列相似性，我们将观察到广泛的增强子活性连续体 这取决于每个增强子的基因组背景和生物化学性质。LTR 5 HS是一个亚类， 人类基因组中最近内源化的逆转录病毒元件HERV-K，并且含有 在人类中是多态的LTR 5 HS元件在人类胚胎中具有转录活性， 作为控制人胚胎癌衍生细胞系中275个基因表达的增强子。我们 将同时和单独地扰乱这些因素，并精确地定义这些因素的影响。 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 将数十种gRNA引入细胞的系统，结合LTR 5 HS的序列相似性， 元件使得与激活或抑制结构域融合的dCas 9能够靶向697个LTR 5 HS中的大多数 人类基因组中的元素。在时间轴上，我们将监测新生转录和长距离转录。 扰动后元素的联系，并推断子网络的活动和合作模式 这取决于这些测量的定时和幅度。其次，我们将创建一个池删除 所有LTR 5 HS元件的文库，并使用单细胞免疫荧光法测量每个元件对新生转录的影响。 从单细胞捕获gRNA序列沿着新生RNA并针对gRNA进行选择技术 对生长、分化和多能性表型有害。这种高粒度的方法将提供 一个将增强子的生物化学性质和基因组背景与转录组学和 表型特性。

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.