Thatavarty Apoorva, Sagy Naor, Erdos Michael R, Lee Isac, Simpson Jared T, Timp Winston, Collins Francis S, Bar Daniel Z
Department of Pediatrics - Division of Infectious Disease, Texas Children's Hospital, Baylor College of Medicine, Houston, TX, USA.
Department of Oral Biology, Goldschleger School of Dental Medicine, Gray Faculty of Medical & Health Sciences, Tel Aviv University, Tel Aviv, Israel.
Epigenetics Chromatin. 2025 Jul 1;18(1):39. doi: 10.1186/s13072-025-00602-9.
Characterization of DNA binding sites for specific proteins is of fundamental importance in molecular biology. It is commonly addressed experimentally by chromatin immunoprecipitation and sequencing (ChIP-seq) of bulk samples (10-10 cells). We have developed an alternative method that uses a Chromatin Antibody-mediated Methylating Protein (ChAMP) composed of a GpC methyltransferase fused to protein G. By tethering ChAMP to a primary antibody directed against the DNA-binding protein of interest, and selectively switching on its enzymatic activity in situ, we generated distinct and identifiable methylation patterns adjacent to the protein binding sites. This method is compatible with methods of single-cell methylation-detection and single molecule methylation identification. Indeed, as every binding event generates multiple nearby methylations, we were able to confidently detect protein binding in long single molecules.
特定蛋白质的DNA结合位点的表征在分子生物学中具有至关重要的意义。通常通过对大量样本（10-10个细胞）进行染色质免疫沉淀和测序（ChIP-seq）来进行实验研究。我们开发了一种替代方法，该方法使用一种由与蛋白G融合的GpC甲基转移酶组成的染色质抗体介导甲基化蛋白（ChAMP）。通过将ChAMP与针对感兴趣的DNA结合蛋白的一抗相连，并在原位选择性地开启其酶活性，我们在蛋白结合位点附近产生了独特且可识别的甲基化模式。该方法与单细胞甲基化检测和单分子甲基化鉴定方法兼容。实际上，由于每个结合事件都会产生多个附近的甲基化，我们能够在长单分子中可靠地检测到蛋白质结合。
