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

项目摘要 顺式调控元件(Cres)已被证明在正确的基因表达程序中起着重要作用 而这些区域的扰动有助于癌症的发展和进展。虽然很多人 监管不当的积极监管因素的后果实例，包括增强剂和 启动子存在，但对干扰的负面调控元件(NRES)的作用知之甚少 例如消音剂和增强剂阻滞剂。最近的证据表明，消音器的数量与 增强子和富含疾病相关的变体，表明这种不平衡不是由于基因组NRE 频率相反，由于缺乏类似于那些的高通量报告分析，NRES的研究不足 加速了增强子识别。目前，在基因组中定位NRES的唯一标准包括分析 这依赖于报告信号的减少或丢失。这会导致较高的假阳性率，因为 合并的高通量分析所固有的伪影可能会导致信号减少。高噪音和低噪音 来自RNA-seq池的信号，其中最强的抑制元件由最少的转录本代表，产生 假阴性率高。这大大增加了准确检测低转录本所需的测序深度 强大的NRES水平。由于这些挑战，NRE检测结果的重复性很差。我们 建议通过开发高通量的报告分析系统来解决这一差距，该系统具有强大的NRES 通过使用dCas9-产生一个阳性信号，观察到报告基因转录的增加 SgRNA系统。我们的dCas9反向记者分析(HT-dCIRA)克服了传统NRE记者的局限性 使用信号反转进行分析，允许稳健可靠地识别消音器和增强剂 人类基因组中的阻断剂。提高可复制性将允许检测差异较大的NRES 在多个样本中活跃，比较驱动正常状态和肿瘤状态的调节元件 或对药物治疗的反应可行。减少了对测序深度的要求和可靠的结果 更少的重复次数将使这项技术适用于广泛的癌细胞系。 这将加快识别癌症状态常见的新型消音剂和增强子阻滞剂 NRES因细胞类型不同而不同。我们将在全基因组筛选中展示我们的分析方法的适用性。 用于MCF-7乳腺癌细胞中的消音剂和增强子阻滞剂。虽然许多研究都检查了 MCF7细胞对17β-雌二醇的反应及雌激素受体1对增强剂的激活作用 (ERα)，对ERα-NRE的关系知之甚少。我们将应用ht-dCIRA来标识这两个系统中的活动NRE 17个经β-雌二醇处理和未处理的MCF-7细胞首次确定沉默和增强子阻滞剂 在ERα反应中以不同方式被激活。

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.