Bray David, Hook Heather, Zhao Rose, Keenan Jessica L, Penvose Ashley, Osayame Yemi, Mohaghegh Nima, Chen Xiaoting, Parameswaran Sreeja, Kottyan Leah C, Weirauch Matthew T, Siggers Trevor
Department of Biology, Boston University, Boston, MA, USA.
Biological Design Center, Boston University, Boston, MA, USA.
Cell Genom. 2022 Feb 9;2(2). doi: 10.1016/j.xgen.2022.100098.
Non-coding DNA variants (NCVs) impact gene expression by altering binding sites for regulatory complexes. New high-throughput methods are needed to characterize the impact of NCVs on regulatory complexes. We developed CASCADE (Customizable Approach to Survey Complex Assembly at DNA Elements), an array-based high-throughput method to profile cofactor (COF) recruitment. CASCADE identifies DNA-bound transcription factor-cofactor (TF-COF) complexes in nuclear extracts and quantifies the impact of NCVs on their binding. We demonstrate CASCADE sensitivity in characterizing condition-specific recruitment of COFs p300 and RBBP5 (MLL subunit) to the promoter in lipopolysaccharide (LPS)-stimulated human macrophages and quantify the impact of all possible NCVs. To demonstrate applicability to NCV screens, we profile TF-COF binding to ~1,700 single-nucleotide polymorphism quantitative trait loci (SNP-QTLs) in human macrophages and identify perturbed ETS domain-containing complexes. CASCADE will facilitate high-throughput testing of molecular mechanisms of NCVs for diverse biological applications.
非编码DNA变异（NCV）通过改变调控复合物的结合位点来影响基因表达。需要新的高通量方法来表征NCV对调控复合物的影响。我们开发了CASCADE（用于在DNA元件处检测复合物组装的可定制方法），这是一种基于阵列的高通量方法，用于分析辅因子（COF）的募集情况。CASCADE可识别核提取物中与DNA结合的转录因子-辅因子（TF-COF）复合物，并量化NCV对其结合的影响。我们展示了CASCADE在表征脂多糖（LPS）刺激的人类巨噬细胞中COF p300和RBBP5（MLL亚基）对启动子的条件特异性募集方面的敏感性，并量化了所有可能的NCV的影响。为了证明其在NCV筛选中的适用性，我们分析了TF-COF与人巨噬细胞中约1700个单核苷酸多态性数量性状位点（SNP-QTL）的结合情况，并鉴定了受干扰的含ETS结构域的复合物。CASCADE将有助于对NCV的分子机制进行高通量测试，以用于各种生物学应用。
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