CASCADE: A high-throughput assay to characterize gene-regulatory complexes affected by single-nucleotide polymorphisms

项目摘要 研究已经确定了数千个单核苷酸多态(SNPs)与一系列 疾病，但原因核苷酸变化和机制在很大程度上仍不清楚。一股水流 该领域的瓶颈是SNP机制的表征大大滞后于SNP的识别。分析 提示潜在疾病风险的因果变异通常是起作用的非编码SNP(NcSNP) 通过它们对基因表达的影响。NcSNPs影响基因表达的主要方式是 通过改变转录因子-辅因子(TF-COF)复合体的结合位点。因此，一位少校 在理解疾病易感性和病因学方面的挑战是描述 数以千计的ncSNP破坏了Tf-COF复合体的结合，从而改变了基因的表达。 为了应对这一挑战，我们最近开发了CASCADE-一种高通量微阵列- 筛选ncSNPs对Tf-COF复合体DNA结合的影响。在这 提议，我们将使用我们的级联方法来研究不同免疫细胞状态下的TF-COF复合体， 并研究与疾病相关的ncSNPs对它们的破坏。要确定细胞状态对我们的 NcSNP注释和Tf-COF复合体，我们将使用细胞系以及原代细胞来检查结果 人类细胞。评估TF类别的COF特异性，以及哪些COF受影响最大 NcSNPs的数量(即，检测覆盖率)，我们将研究包含不同COF和 COF子组件。我们的目标是将CASCADE开发为通用的高吞吐量平台，以 对受ncSNPs影响的调控复合体进行生物物理学表征。

Project Summary Studies have identified thousands of single-nucleotide polymorphisms (SNPs) associated with a range of diseases, but the causal nucleotide changes and mechanisms remain largely unknown. A current bottleneck in the field is that characterization of SNP mechanism greatly lags SNP identification. Analyses suggest that causal variants underlying disease risk are often non-coding SNP (ncSNPs) that function through their effects on gene expression. The primary means by which ncSNPs affect gene expression is by altering binding sites for transcription factor-cofactor (TF-CoF) complexes. Therefore, a major challenge in understanding disease susceptibility and etiology is to characterize the mechanisms by which the thousands of ncSNPs disrupt the binding of TF-CoF complexes to alter gene expression. To address this challenge, we have recently developed CASCADE – a high-throughput microarray- based method to screen the impact of ncSNPs on the DNA-binding of TF-CoF complexes. In this proposal, we will use our CASCADE approach to study TF-CoF complexes in diverse immune cell states, and study their disruption by disease-associated ncSNPs. To determine the cell state-dependence of our ncSNP annotations and TF-CoF complexes, we will examine results using cell lines as well as primary human cells. To assess CoF specificity for TF classes, and which CoFs are affected by the largest number of ncSNPs (i.e., assay coverage), we will study TF-CoF complexes comprising diverse CoFs and CoF subcomponents. Our goal is to develop CASCADE as a general high-throughput platform to biophysically characterize the regulatory complexes affected by ncSNPs.