Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences): a novel strategy for high-resolution mapping of DNA binding sites

DNA and protein interactions are essential for cellular processes and have major implications in cancer biology. DNA binding proteins, particularly transcription factors (TF), are crucial in regulating gene expression and downstream protein functions. Studying the intricate DNA-protein interactions helps unravel specific functions of TFs that are involved in cancer pathogenesis, identify regulatory pathways, and ultimately uncover therapeutic targets. Currently, the lack of high-throughput screening techniques makes research in cancer associated-TFs challenging. Therefore, we have developed an innovative Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences). Here, K562 tumor cells were coencapsulated with NK92 and antibody-conjugated (NFkB/RelA) magnetic beads in a microfluidic droplet-based system. After NK92 immune cells killed the K562 target cells, we collected RelA-bound DNA from the dead cells and analyzed DNA-binding sites using ChIP-seq. The results reveal the RelA genome-wide association study (GWAS) of the target K562 tumor cells in response to immune cell killing. The genome-wide mapping matched the expected profile of NFkB, validating the ability of our Iso-CUT&MAP method to accurately identify NFkB binding in response to NK92 killing of K562. This novel technique holds promise for precision medicine in understanding apoptosis biology and the influence of immune cell or chemotherapy-induced tumor cell killings. Overall design: In this study, we have developed a novel single-cell method, named Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences), to investigate DNA-protein interactions and evaluate the cytotoxicity of immune cells towards target tumor cells. This innovative method combines droplet microfluidics and imaging-based technology to facilitate the isolation and analysis of protein-bound DNA from individual apoptotic tumor cells. In this approach, an effector cell is paired with a target cell at the single-cell level and magnetic beads, conjugated to antibodies to isolate the protein-bound DNA from the apoptotic target cell in droplets (Figure 1). Subsequently, the beads are released from droplets, and the genetic material captured and subjected to sequencing using ChIP-seq.