Zicong Zhang, Ilias Georgakopoulos-Soares, Guillaume Bourque, Nadav Ahituv, Fumitaka Inoue
Using various biochemical assays that identify transcription factor (TF) binding and histone modifications, cis-regulatory elements (CREs) can be annotated in a genome-wide manner. However, these assays are descriptive and require functional validation. To the best of our knowledge, no technology can simultaneously analyze the regulatory function and epigenomic modifications of a specific sequence. Here, we develop an enrichment followed by epigenomic profiling massively parallel reporter assay (e2MPRA). This technique uses lentivirus to enrich for the integration of specific CREs into the genome and applies MPRA, Cut&Tag or ATAC-seq on them enabling simultaneous, high-throughput analysis of regulatory activity, protein binding, and epigenetic modification. We demonstrate that e2MPRA can dissect the epigenetic functions of TF motifs arranged within synthetic enhancers and evaluate the effects of sequence perturbation on epigenetic states. In summary, e2MPRA advances our understanding of the regulatory code, its effect on the epigenome and how its alteration leads to phenotypic effects.
Zicong Zhang, Ilias Georgakopoulos-Soares, Guillaume Bourque, Nadav Ahituv, Fumitaka Inoue. Simultaneous epigenomic profiling and regulatory activity measurement using e2MPRA. 2026-01-14. https://doi.org/10.1038/s41467-026-68422-3
A 100-bp enhancer perturbation library in WTC11 human iPSCs tested single-nucleotide substitutions and two randomized 6-bp sliding-window perturbations per position across selected pluripotency-associated CREs, with integrated lentiMPRA and targeted ATAC-seq/H3K27ac CUT&Tag.
A 400-element pilot library of 100-bp genomic and synthetic CREs was assayed in HepG2 cells using integrated lentiMPRA, with targeted ATAC-seq and H3K27ac CUT&Tag measurements on the same integrated library.
A synthetic enhancer library arranged nine liver-expressed TF motifs in three combinatorial classes on two neutral templates, with 200 pilot-derived controls, and assayed it in HepG2 cells by integrated lentiMPRA plus targeted ATAC-seq and H3K27ac CUT&Tag.
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