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Gene regulation is mediated by site-specific DNA-binding proteins or transcription factors (TFs), which form protein complexes at regulatory loci either to activate or repress the expression of a target gene. The study of the dynamic properties of these regulatory DNA-binding complexes has so far been dominated by protein-centered methodologies, aiming to characterize the DNA-binding behavior of one specific protein at a time. With the emerging evidence for a role of DNA in allosterically influencing DNA-binding protein complex formation, there is renewed interest in DNA-centered approaches to capture protein complexes on defined regulatory loci and to correlate changes in their composition with alterations in target gene expression. In this review, we present the current state-of-the-art in such DNA-centered approaches and evaluate recent technological improvements in the purification as well as in the identification of regulatory DNA-binding protein complexes within or outside their biological context. Finally, we suggest possible areas of improvement and assess the putative impact of DNA-centered methodologies on the gene regulation field for the forthcoming years.

DNA-centered approaches to characterize regulatory protein–DNA interaction complexes