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bioRxiv · 10.1101/2025.04.21.649048

Long-range massively parallel reporter assay reveals rules of distal enhancer-promoter interactions

Wu, Y.·Li, J.·Bartley-Dier, E. L.·Pitts, C.·Cohen, B. A.
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Abstract

Long-range activation is an essential property of enhancers, yet the features determining long-range enhancer activities have not been systematically investigated due to a lack of high-throughput methods to measure long-range enhancer activities efficiently. To address this gap, we present a long-range massively parallel reporter assay (long-range MPRA), an assay allowing the measurement of hundreds of enhancers at multiple distances from a genome-integrated promoter. The long-range MPRA assay features two independent landing pads, allowing modular control over the genome-integrated promoter and enhancer libraries. We showcased the capability of long-range MPRA by testing over 300 K562 enhancers, as well as a set of enhancer combinations, at distances up to 100 kb. We found that enhancers long-range activities are primarily determined by their intrinsic strength, with strong enhancers retaining more activity over long distances, while weak enhancers rapidly lose activity. Additionally, we found that GATA1-bound enhancers are more resistant to distance-dependent loss of activity, suggesting that TF binding also modulates long-range function. Finally, testing long-range enhancer activities with three different promoters (HBE, HBG and GAPDH) revealed that long-range enhancer-promoter interactions rely on not only enhancer properties but also promoter responsiveness.

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Wu, Y., Li, J., Bartley-Dier, E. L., Pitts, C., Cohen, B. A.. 2025-04-22. Long-range massively parallel reporter assay reveals rules of distal enhancer-promoter interactions. https://doi.org/10.1101/2025.04.21.649048

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