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Massively parallel characterization of transcriptional regulatory elements

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Agarwal, Vikram, Inoue, Fumitaka, Schubach, Max, Penzar, Dmitry, Martin, Beth K., Dash, Pyaree Mohan, Keukeleire, Pia, Zhang, Zicong +9 more·2025·Nature
DOI 10.1038/s41586-024-08430-9PMC PMC11903340

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Abstract

The human genome contains millions of candidate cis -regulatory elements (cCREs) with cell-type-specific activities that shape both health and many disease states 1 . However, we lack a functional understanding of the sequence features that control the activity and cell-type-specific features of these cCREs. Here we used lentivirus-based massively parallel reporter assays (lentiMPRAs) to test the regulatory activity of more than 680,000 sequences, representing an extensive set of annotated cCREs among three cell types (HepG2, K562 and WTC11), and found that 41.7% of these sequences were active. By testing sequences in both orientations, we find promoters to have strand-orientation biases and their 200-nucleotide cores to function as non-cell-type-specific ‘on switches’ that provide similar expression levels to their associated gene. By contrast, enhancers have weaker orientation biases, but increased tissue-specific characteristics. Utilizing our lentiMPRA data, we develop sequence-based models to predict cCRE function and variant effects with high accuracy, delineate regulatory motifs and model their combinatorial effects. Testing a lentiMPRA library encompassing 60,000 cCREs in all three cell types further identified factors that determine cell-type specificity. Collectively, our work provides an extensive catalogue of functional CREs in three widely used cell lines and showcases how large-scale functional measurements can be used to dissect regulatory grammar.

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  • In K562 cells, activators usually dominate, and repressive interactions are scarce.

    “In a human leukemia cell line commonly used for MPRA studies42,43 (K562), by contrast, activators usually dominate, and repressive interactions are scarce.”

    From Design principles of cell-state-specific enhancers in hematopoiesis (2025) · cites this paper as [42]

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Cited by

Sorted by citations

Authors
Agarwal, V., Inoue, F., Schubach, M., Penzar, D., Martin, B. K., Dash, P. M., Keukeleire, P., Zhang, Z., Sohota, A., Zhao, J., Georgakopoulos-Soares, I., Noble, W. S., Yardımcı, G. G., Kulakovskiy, I. V., Kircher, M., Shendure, J., & Ahituv, N.
Affiliations
  • University of Washington (US)
  • University of California, San Francisco (US)
  • Berlin Institute of Health at Charité - Universitätsmedizin Berlin (DE)
  • Russian Academy of Sciences (RU)
  • University of Lübeck (DE)
  • Kyoto University (JP)
  • Oregon Health & Science University (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
  • Agarwal, Vikram
  • Shendure, Jay
  • Ahituv, Nadav
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2025-01-15
Volume
639
Issue
8054
Pages
411-420
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-024-08430-9
PMC
PMC11903340
OpenAlex
W4406385639
Cited by
146
References
19
Citations by year
2026: 69 · 2025: 49 · 2024: 20 · 2023: 8
FWCI
35.77
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nd)
  • RePEc: Research Papers in Economics (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
Funders
  • Howard Hughes Medical Institute
  • Deutsche Forschungsgemeinschaft (464313370)
  • Ministry of Education, Culture, Sports, Science and Technology
  • Russian Science Foundation (20–74-10075)
  • National Institutes of Health
  • Japan Society for the Promotion of Science (24K02004, 24K18101)
  • National Heart, Lung, and Blood Institute (T32HL007093)
  • National Human Genome Research Institute (HG009446, UM1HG009408, U24HG009446, UM1HG011966)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK124769, P30DK098722)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
Keywords
cis-regulatory elements, enhancers, promoter, cell type specificity, massively parallel reporter assay, combinatorial transcriptional regulation, variant effect prediction, HepG2 cells, K562 cells

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