A Chemical Epigenetic Probe to Capture the Site-Specific DNA-Binding Protein Complex

Site-specific DNA binding by proteins is critical for regulating genetic activity and cell fate decision. However, identifying proteins bound to specific genomic regions (e.g., promoter or enhancer) remains challenging. To address this, we developed a chemical epigenetic tool, named Site-specific non-canonical amino acid resolved Protein EnRichment (SUPER) system, incorporating a photo-crosslinking amino acid into nuclease-deficient dCas9 mutant. Human pluripotent stem cells (hPSCs) carrying SUPER enables the capture of proteins bound to, in theory, any genomic location, facilitating the study of the cell context-dependent DNA-protein interactions. Using SUPER, we identified OCT4/SOX2/CARHSP1 complex binding to the NANOG promoter to maintain pluripotency in hPSCs. During ectoderm differentiation, ZIC2 acts as a competitive inhibitor, binding the same promoter to downregulate NANOG expression and promote differentiation. Additionally, SUPER identified ZNF8 binding to the distal regulatory region of OCT4 and maintain naïve pluripotency. In summary, SUPER provides a robust system for uncovering the cell context-dependent, site-specific genome regulators, offering valuable insights into gene regulation networks driving cell fate transitions.