High-throughput inverted reporter assay for characterization of silencers and enhancer blockers

项目摘要 顺式调控元件（Cis-regulatory elements，克雷斯）在基因的正常表达过程中起着重要作用 并且这些区域的扰动有助于癌症的发展和进展。虽然许多 错误调节的正调节元件，包括增强子和 启动子，存在，相对较少的是已知的破坏负调控元件（NREs）的作用， 例如消音剂和增强剂阻断剂。最近的证据表明，消音器是丰富的， 增强子和疾病相关的变异富集，表明这种不平衡不是由于基因组NRE 频率.相反，NRE由于缺乏类似于那些用于检测NRE的高通量报告基因测定法而未被充分研究。 加速增强子鉴定。目前，在基因组中定位NRE的唯一标准涉及测定 其依赖于报告信号的减少或丢失。这导致高假阳性率，由于多重 合并的高通量测定固有的伪影，其可导致信号降低。高噪音低噪音 来自RNA-seq池的信号，其中强阻遏元件由最少的转录物代表，产生 高假阴性率。这极大地增加了准确检测低转录物所需的测序深度。 强大的NREs。由于这些挑战，NRE测定结果的可重复性一直很差。我们 我建议通过开发一种高通量报告基因测定系统来解决这一差距， 产生阳性信号，观察到报告基因转录增加，通过使用dCas 9- sgRNA系统。我们的dCas 9反向报告基因测定（ht-dCIRA）克服了传统NRE报告基因的局限性， 使用信号反转的测定，允许稳健和可靠地鉴定沉默剂和增强剂 人类基因组中的阻断剂。提高可复制性将允许检测NRE， 在多个样本中有效，比较驱动正常与肿瘤状态的调节元件 或对药物治疗的反应可行。降低测序深度要求并获得可靠结果， 更少的重复将另外使该技术适用于广泛的癌细胞系。 这将加快识别新的沉默剂和增强剂阻断剂，这些是癌症状态与 不同细胞类型的NRE。我们将证明我们的检测方法在全基因组筛选中的适用性 用于MCF-7乳腺癌细胞中的沉默剂和增强剂阻断剂。虽然许多研究已经检查了 MCF-7细胞对17β-雌二醇的反应及雌激素受体1对增强子的激活作用 (ERα），对ERα-NRE关系知之甚少。我们将应用ht-dCIRA来识别这两种情况下的活性NRE。 17β-雌二醇处理和未处理的MCF-7细胞，以首次确定沉默剂和增强剂阻断剂 在ERα反应中被不同激活。

Project Summary Cis-regulatory elements (CREs) have been shown to play a significant role in proper gene expression programs and perturbations of these regions contribute to the development and progression of cancer. While many examples of the consequences of misregulated positive regulatory elements, including enhancers and promoters, exist, comparatively little is known about the role of disrupted negative regulatory elements (NREs) such as silencers and enhancer blockers. Recent evidence suggests that silencers are as abundant as enhancers and enriched for disease associated variants, indicating this imbalance is not due to genomic NRE frequency. Rather, NREs are understudied due to lack of a high-throughput reporter assay similar to those that accelerated enhancer identification. Currently the only standard for locating NREs in the genome involves assays that rely on a reduction or loss of reporter signal. This results in high false positive rates due to the multiple artefacts inherent to pooled high-throughput assays which can cause reduction in signal. High noise and low signal from RNA-seq pools, where strong repressive elements are represented by the fewest transcripts, produce high false negative rates. This vastly increases the sequencing depth needed to accurately detect low transcript levels of potent NREs. As a result of these challenges, replicability of NRE assay results have been poor. We propose to address this gap by developing a high-throughput reporter assay system where strong NREs generate a positive signal, observed as an increase in reporter gene transcription, through the use of a dCas9- sgRNA system. Our dCas9 Inverted Reporter Assay (ht-dCIRA) overcomes limitations of traditional NRE reporter assays with the use of signal inversion, allowing for robust and reliable identification of silencers and enhancer blockers in the human genome. Improving replicability will allow the detection of NREs that are differentially active across multiple samples, making comparisons of the regulatory elements driving normal vs tumor states or response to drug treatment feasible. Reduction of sequencing depth requirements and reliable results with fewer replicates will additionally make this technology applicable for use across a broad set of cancer cell lines. This will expedite identification of novel silencers and enhancer blockers that are common to cancer states versus NREs that vary among cell types. We will demonstrate the applicability of our assay in a genome-wide screen for silencers and enhancer blockers in MCF-7 breast cancer cells. While many studies have examined the response of MCF-7 cells to 17β-estradiol and subsequent activation of enhancers by estrogen receptor one (ERα), little is known about the ERα-NRE relationship. We will apply ht-dCIRA to identify active NREs in both 17β-estradiol treated and untreated MCF-7 cells to determine for the first time silencers and enhancer blockers that are differentially activated in the ERα response.