Simultaneous epigenomic profiling and regulatory activity measurement using e2MPRA.

Area of Science:

• Genomics and Molecular Biology
• Epigenetics
• Regulatory Genomics

Background:

• Cis-regulatory elements (CREs) significantly impact phenotypes and diseases.
• Current methods like ChIP-seq, Cut&Tag, and ATAC-seq identify CREs but are descriptive.
• High-throughput technologies like MPRAs are needed to assess functional activity and variant effects.

Purpose of the Study:

• To develop a novel technology enabling simultaneous analysis of CRE regulatory function, TF/cofactor binding, and epigenomic modifications.
• To overcome the limitations of existing methods that cannot integrate functional and epigenomic profiling.
• To provide a high-throughput platform for dissecting the regulatory code and its role in disease.

Main Methods:

• Developed enrichment followed by epigenomic profiling MPRA (e2MPRA), a lentivirus-based MPRA technology.
• Enriched specific CREs for genomic integration, followed by targeted Cut&Tag or ATAC-seq.
• Enabled simultaneous high-throughput analysis of regulatory activity, protein binding, and epigenetic modifications for thousands of CREs and variants.

Main Results:

• Demonstrated e2MPRA's capability to dissect epigenetic functions of TF motifs in synthetic enhancers.
• Showcased the analysis of enhancer sequence variant effects on epigenetic modifications.
• Validated the simultaneous assessment of regulatory activity, protein binding, and epigenomic data for candidate CREs.

Conclusions:

• e2MPRA is a novel technology for simultaneous high-throughput analysis of CRE function, protein interactions, and epigenomic states.
• This method enhances understanding of the regulatory code, its impact on the epigenome, and disease mechanisms.
• e2MPRA provides a powerful tool for investigating how alterations in the regulatory code contribute to human diseases.