A systematic guide for identifying transcription factors that directly regulate the expression of a gene of interest

Transcriptional regulation lies at the heart of cellular identity and function, hinging on the precise binding of transcription factors (TFs) and cofactors to gene regulatory elements such as promoters and enhancers. Although it is relatively routine to profile genome-wide DNA binding landscapes of proteins, identifying the specific proteins that bind to, and regulate the transcription of, a particular gene of interest (GOI) remains a persistent experimental and conceptual challenge. This gene-centric question is complicated by the multilayered regulatory environment in which each gene resides, comprising 3D chromatin structure, enhancer-promoter looping, DNA accessibility, histone modifications, and cell state-dependent protein dynamics. In this review, we dissect the strengths, limitations, and biological relevance of current approaches for studying direct protein-DNA interactions, distinguishing between protein-centric and DNA-centric methodologies. We introduce a conceptual matrix of biological relevance, integrating the origin of DNA and protein elements (<i>cis</i> and <i>trans</i>) to evaluate false-positive and false-negative risks across experimental systems. Moreover, we explore how perturbation strategies-gain and loss of function-can complement steady-state profiling to establish causality in gene regulation. By critically examining both established tools and emerging techniques such as genome editing, synthetic chromosomes, and high-resolution imaging, we provide a practical framework for investigators seeking to uncover direct regulators of specific genes. Our goal is to guide the design of experiments that balance biological relevance, sensitivity, and interpretability to ultimately answer a deceptively simple question: What TFs directly regulate the expression of my GOI?