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

Area of Science:

• Genomics
• Molecular Biology
• Epigenetics

Background:

• Chromatin regulatory factors (CRFs) control essential genomic functions.
• Interactions between CRFs are poorly understood due to experimental limitations.
• Existing methods struggle to analyze multiple CRFs and their interactions simultaneously.

Purpose of the Study:

• To develop an advanced technology for robust detection of co-localization events among CRFs.
• To enable comprehensive pairwise analysis of epigenetic modifiers and transcription factors.
• To provide unprecedented resolution for studying chromatin dynamics and gene regulation.

Main Methods:

• Development of Hi-Plex CUT&Tag technology.
• Utilizing 36 barcoded monoclonal antibodies (mAbs) for detecting over 600 CRF pairs.
• Analyzing Tn5 tagmentation fragments for co-localized events, genomic sequences, and molecular distances.

Main Results:

• Robust detection of co-localization events across >600 CRF pairs with minimized background.
• Comprehensive pairwise analysis of histone post-translational modifications (PTMs), writers, and transcription factors (TFs).
• Identification of novel bivalent events and epigenetic-context-dependent transcriptional regulation in a single experiment.
• Single-cell analysis of synergistic interactions between CRF pairs.

Conclusions:

• Hi-Plex CUT&Tag overcomes limitations of previous methods for studying CRF interactions.
• The technology enables the detection of novel biological interactions impacting gene regulation.
• Single-cell Hi-Plex CUT&Tag offers high-resolution insights into chromatin dynamics.