Simultaneous epigenomic profiling and regulatory activity measurement using e2MPRA

Area of Science:

• Genomics
• Epigenetics
• Molecular Biology

Background:

• Genome-wide annotation of cis-regulatory elements (CREs) relies on descriptive assays for transcription factor (TF) binding and histone modifications.
• Functional validation of CREs is essential but challenging, with no current technology enabling simultaneous analysis of regulatory function and epigenomic state.

Purpose of the Study:

• To develop a novel technology for simultaneous, high-throughput analysis of CRE regulatory function and associated epigenomic modifications.
• To enable functional dissection of TF motifs within synthetic enhancers and assess sequence perturbation effects on epigenetic states.

Main Methods:

• Development of an enrichment followed by epigenomic profiling massively parallel reporter assay (e2MPRA).
• Utilizes lentivirus for CRE enrichment and integration, coupled with MPRA, Cut&Tag, or ATAC-seq.
• Enables simultaneous assessment of regulatory activity, protein binding, and epigenetic modifications.

Main Results:

• Demonstrated e2MPRA's capability to dissect epigenetic functions of TF motifs in synthetic enhancers.
• Successfully evaluated the impact of sequence perturbations on epigenetic states using e2MPRA.
• Achieved simultaneous, high-throughput analysis of regulatory function and epigenomic modifications for specific sequences.

Conclusions:

• e2MPRA is a powerful new technique for comprehensive analysis of cis-regulatory elements.
• The method advances understanding of the regulatory code, its impact on the epigenome, and phenotypic consequences of alterations.