Protocol to capture transcription factor-mediated 3D chromatin interactions using affinity tag-based BL-HiChIP

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Pioneer transcription factors (TFs) play a crucial role in establishing higher-order chromatin structures that direct gene transcription.
• Understanding TF-mediated 3D chromatin interactions is essential for deciphering gene regulatory mechanisms.
• Existing methods may have limitations in capturing interactions for specific TFs without optimal antibodies.

Purpose of the Study:

• To present a detailed protocol for capturing TF-mediated 3D chromatin interactions.
• To enable the analysis of specific TF functions in genome organization and gene regulation.
• To provide a versatile method applicable across different cell types and for various TFs.

Main Methods:

• Development and description of the affinity tag-based bridge linker (BL)-Hi-chromatin immunoprecipitation (HiChIP) protocol.
• Detailed steps for constructing FLAG-tagged TFs.
• Procedures for library preparation, sequencing, data analysis, and quality control of BL-HiChIP experiments.

Main Results:

• Successful implementation of the BL-HiChIP protocol for capturing TF-mediated 3D chromatin interactions.
• Demonstration of the protocol's applicability in analyzing specific TF binding and its role in chromatin architecture.
• Validation of the method's robustness through sequencing, data analysis, and quality control procedures.

Conclusions:

• The BL-HiChIP protocol offers a powerful tool for investigating TF-driven 3D genome organization.
• This method facilitates the study of specific TFs without reliance on high-quality antibodies.
• The protocol has broad potential applications in 3D chromatin analysis across diverse biological systems.