CASCADE - Customizable high-throughput platform for profiling cofactor recruitment to DNA to characterize cis-regulatory elements and screen non-coding SNPs

Determining how DNA variants affect the binding of regulatory complexes to cis-regulatory elements (CREs) and non-coding single-nucleotide polymorphisms (ncSNPs) is a challenge in genomics. To address this challenge, we present CASCADE (Comprehensive ASsessment of Complex Assembly at DNA Elements) – a protein-binding microarray (PBM)-based approach to profile the recruitment of cofactors (COFs) to DNA sequence variants, and to infer the identity of the transcription factor-cofactor (TF-COF) complexes involved. We use CASCADE to characterize regulatory complexes binding to CREs and SNP quantitative trait loci (SNP-QTLs) in resting and stimulated human macrophages. By profiling p300 we correctly identify IRF3 and NF-κB as LPS-dependent regulators of the chemokine CXCL10, and by profiling a set of five functionally diverse COFs we identify a prevalence of ETS sites mediating COF recruitment at SNP-QTLs in macrophages. Our results demonstrate that CASCADE is a customizable, high-throughput platform to link DNA variants with the biophysical complexes that mediate functions such as chromatin modification or remodeling in a cell state-specific manner. LPS-responsive CXCL10 promoter segment CASCADE (ID: 085605) - These experiments profile recruitment of transcriptional cofactors (COFs: p300 and RBBP5) as well as transcription factors (TFs: IRF3, p65, IRF2, IRF8) to an LPS-responsive segment of the CXCL10 promoter using tiling probes and all single variant probes of each tile. Recruitment of COFs and TFs was profiled directly from nuclear extracts collected from untreated (UT) or LPS-stimulated (LPS45) PMA-treated THP-1 cells. Differential COF recruitment screen (ID: 085920) – These experiments profile recruitment of a panel of COFs (p300, SMARCA4, TBL1XR1, RBBP5, HDAC1, and GCN5) as well as TFs (PU.1 and p65) to pairs of reference and variant alleles from previously characterized myeloid SNP-QTLs. Technical replicates were performed (REP1 and REP2) for each COF/TF screened. SNP-QTL CASCADE (ID: 086248) – These experiments employ the full CASCADE approach to characterize recruitment motifs for significant differential COF recruitment and TF binding events measured using the previous COF recruitment screen. The same set of COFs/TFs were profiled in this series of experiments (p300, SMARCA4, TBL1XR1, GCN5, RBBP5, HDAC1, PU.1, and p65).