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

用于表征沉默子和增强子阻断剂的高通量反向报告基因测定

基本信息

批准号：
10578838
负责人：
Alan P Boyle
金额：
$ 20.74万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-23 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10578838
关键词：
Acceleration Address Automobile Driving Biological Assay Breast Cancer Cell Cancer Biology Cancer cell line Cell Line Chromatin Clustered Regularly Interspaced Short Palindromic Repeats DNA Sequence Alteration Data Data Set Detection Development Disease Disparity Elements Enhancers Estradiol Estrogen Receptor alpha Estrogen Receptors Frequencies Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome Genomics Goals Human Genome Lead Libraries MCF7 cell Malignant Neoplasms Maps Methods Modeling Morphologic artifacts Mutation Noise Normal Cell Oncogenes Outcome Performance Pharmaceutical Preparations Pharmacotherapy Plasmids Play Publishing Regulatory Element Reliability of Results Reporter Reporter Genes Repression Reproducibility Research Role Sampling Signal Transduction Single Nucleotide Polymorphism System Technology Testing Time Transcript Transfection Untranslated RNA Variant Work cancer cell carcinogenesis cell type comparative design detection assay genome wide screen high throughput screening improved innovation neoplastic cell novel programs promoter response transcription factor transcriptome sequencing tumor tumor progression tumorigenesis variant of unknown significance

项目摘要

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.

项目概要顺式调控元件 (CRE) 已被证明在正确的基因表达程序中发挥着重要作用这些区域的扰动会导致癌症的发生和进展。积极监管因素（包括增强剂和增强剂）监管不当造成的后果的例子启动子存在，但人们对破坏的负调控元件（NRE）的作用知之甚少例如消音器和增强剂阻断剂最近的证据表明消音器的数量与增强子和富集疾病相关变异，表明这种不平衡不是由于基因组 NRE 造成的相反，由于缺乏类似于那些的高通量报告分析，NRE 的研究不足。目前，在基因组分析中定位 NRE 的唯一标准涉及加速增强子识别。依赖于报告信号的减少或丢失，这会导致由于多重性而导致高假阳性率。合并高通量检测固有的伪影可能会导致信号降低。来自RNA-seq池的信号，其中强抑制元件由最少的转录本代表，产生高假阴性率，这大大增加了准确检测低转录本所需的测序深度。由于这些挑战，NRE 测定结果的可重复性很差。建议通过开发高通量报告分析系统来解决这一差距，其中强大的 NRE 通过使用 dCas9- 产生阳性信号，观察到报告基因转录增加我们的 sgRNA 系统。我们的 dCas9 反向报告基因检测 (ht-dCIRA) 克服了传统 NRE 报告基因的局限性。使用信号反转进行测定，可以稳健可靠地识别沉默子和增强子提高人类基因组中的阻断剂将允许检测差异性的 NRE。在多个样本中活跃，比较驱动正常状态与肿瘤状态的调控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的结果。更少的重复将使该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沉默剂和增强剂阻滞剂 NRE 因细胞类型而异。我们将证明我们的检测方法在全基因组筛选中的适用性。虽然许多研究已经检验了 MCF-7 乳腺癌细胞中的沉默剂和增强剂阻滞剂。 MCF-7 细胞对 17β-雌二醇的反应以及雌激素受体 1 增强子的随后激活 (ERα)，关于 ERα-NRE 关系知之甚少，我们将应用 ht-dCIRA 来识别两者中的活性 NRE。 17β-雌二醇处理和未处理的 MCF-7 细胞首次确定沉默剂和增强剂阻滞剂它们在 ERα 反应中被不同程度地激活。