Global Mapping of Combinatorial Chromatin Regulatory Events Using Hi-Plex CUT&Tag

Epigenetic modulators, transcription factors, and chromatin-associated proteins collaboratively regulate essential genomic functions, such as transcription, repression, and DNA damage repair. However, the intricate cross-talk or the interaction between these chromatin regulatory factors (CRFs) remains poorly understood, largely due to limitations in experimental methods, which traditionally interrogate the genomic locations of only one CRF. Although the latest methods can study multiple CRFs simultaneously, they cannot examine the interaction between these factors. Inferring biological interactions often requires integrating data from multiple experiments, which can be labor-intensive and imprecise since the measurement is not from the same DNA molecule. To address these challenges, we developed Hi-Plex CUT&Tag, an advanced technology that enables the robust detection of co-localization events across over 600 pairs of CRFs using 36 barcoded monoclonal antibodies (mAbs), while minimizing background signals and cross-contamination. Hi-Plex CUT&Tag facilitates comprehensive pairwise analysis of epigenetic modifiers, including histone post-translational modifications (PTMs), writers, and transcription factors (TFs). Each Tn5 tagmentation fragment provides detailed insights, capturing the identities of two co-localized events, the underlying genomic sequences, and the molecular distance between them. For the first time, numerous novel bivalent events, epigenetic-context-dependent transcriptional regulation, and specific chromatin mark combinations with significant impacts on gene regulation can be detected in a single experiment. Furthermore, single-cell Hi-Plex CUT&Tag enables the analysis of synergistic interactions between CRF pairs at the single-cell level, providing unprecedented resolution for studying chromatin dynamics.