An Integrated High-Throughput Proteocistromic Framework for Mapping the Interactome and Targetome of Human Transcription Factors.

Transcription factors (TFs) are the main regulators of gene expression, controlling diverse cellular processes such as development, differentiation, and disease progression. A comprehensive analysis of their interactomes and targetomes provides valuable insights into their functions, regulatory mechanisms, and context-dependent roles. This chapter presents a robust methodological framework for investigating the interactome and targetome of TFs, applicable to a wide range of TF families. This workflow integrates advanced proteomic approaches-affinity purification mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID)-with the cistromic method, chromatin immunoprecipitation sequencing (ChIP-Seq), to identify both stable and transient protein-protein interactions (PPIs) and to identify interactions with DNA (e.g., DNA-binding targets of TFs). The workflow involves the generation of inducible cell lines expressing each TF under study and performing three parallel proteocistromic experiments (AP-MS, BioID, and ChIP-Seq) along with data analysis for network visualization and functional annotation. The workflow is adaptable to various TF families and experimental contexts, offering a versatile platform for uncovering the dynamic regulatory networks that TFs mediate, on both medium and large scales. This workflow advances our ability to comprehensively characterize TF functions, facilitating a deeper understanding of gene regulation and its implications in both health and disease.