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Cited by 315Genomics and Chromatin DynamicsChromatin Remodeling and CancerEpigenetics and DNA Methylation

Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay

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Kheradpour, Pouya, Ernst, Jason, Melnikov, Alexandre, Rogov, Peter, Wang, Li, Zhang, Xiaolan, Alston, Jessica, Mikkelsen, Tarjei S. +1 more·2013·Genome Research
DOI 10.1101/gr.144899.112PMC PMC3638136
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Abstract

Genome-wide chromatin annotations have permitted the mapping of putative regulatory elements across multiple human cell types. However, their experimental dissection by directed regulatory motif disruption has remained unfeasible at the genome scale. Here, we use a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145-bp DNA segments centered on evolutionarily conserved regulatory motif instances within enhancer chromatin states. We select five predicted activators (HNF1, HNF4, FOXA, GATA, NFE2L2) and two predicted repressors (GFI1, ZFP161) and measure reporter expression in erythroleukemia (K562) and liver carcinoma (HepG2) cell lines. We test 2104 wild-type sequences and 3314 engineered enhancer variants containing targeted motif disruptions, each using 10 barcode tags and two replicates. The resulting data strongly confirm the enhancer activity and cell-type specificity of enhancer chromatin states, the ability of 145-bp segments to recapitulate both, the necessary role of regulatory motifs in enhancer function, and the complementary roles of activator and repressor motifs. We find statistically robust evidence that (1) disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are predictive of enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually inactive. Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale manipulation and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to develop predictive models of gene expression.

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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 [43]

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Cited by

Sorted by citations

Authors
Kheradpour, P., Ernst, J., Melnikov, A., Rogov, P., Wang, L., Zhang, X., Alston, J., Mikkelsen, T. S., & Kellis, M.
Affiliations
Broad Institute (US)
Corresponding author
Kellis, Manolis
ORCID
Venue
Genome Research
Publisher
Cold Spring Harbor Laboratory
Published
2013-03-19
Volume
23
Issue
5
Pages
800-811
ISSN
1088-9051 (ISSN-L)
DOI
10.1101/gr.144899.112
PMC
PMC3638136
OpenAlex
W2159664283
Cited by
315
References
6
Citations by year
2026: 6 · 2025: 18 · 2024: 20 · 2023: 24 · 2022: 38 · 2021: 21 · 2020: 25 · 2019: 25 · 2018: 22 · 2017: 29 · 2016: 22 · 2015: 35 · 2014: 25 · 2013: 5
Type
posted content
Language
en
Open access
Licence
other-oa
Locations
  • Genome Research (publishedVersion, other-oa)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
  • HighWire Press Open Archive (submittedVersion)
Funders
  • Broad Institute
  • National Institutes of Health
  • Harvard Stem Cell Institute
  • National Human Genome Research Institute (HG004037-S1, HG004037, R01HG004037, R01HG006785)
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 › Epigenetics and DNA Methylation
Keywords
enhancers, regulatory motifs, cis-regulatory elements, massively parallel reporter assay, enhancer activity, HNF1, HNF4, FoxA, GATA, Nrf2, GFI1, evolutionary conservation, cell type specificity, K562 cells, HepG2 cells, gene expression prediction
MeSH terms
Base Sequence, Binding Sites, Cells, Chromatin, Chromosome Mapping, Enhancer Elements, Genetic, Gene Expression Regulation, Humans, Promoter Regions, Genetic, Transcription, Genetic, Genome, Human, Conserved Sequence, Genes, Reporter, Hep G2 Cells, Nucleotide Motifs

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