High-throughput functional testing of ENCODE segmentation predictions
Open accessAbstract
The histone modification state of genomic regions is hypothesized to reflect the regulatory activity of the underlying genomic DNA. Based on this hypothesis, the ENCODE Project Consortium measured the status of multiple histone modifications across the genome in several cell types and used these data to segment the genome into regions with different predicted regulatory activities. We measured the cis-regulatory activity of more than 2000 of these predictions in the K562 leukemia cell line. We tested genomic segments predicted to be Enhancers, Weak Enhancers, or Repressed elements in K562 cells, along with other sequences predicted to be Enhancers specific to the H1 human embryonic stem cell line (H1-hESC). Both Enhancer and Weak Enhancer sequences in K562 cells were more active than negative controls, although surprisingly, Weak Enhancer segmentations drove expression higher than did Enhancer segmentations. Lower levels of the covalent histone modifications H3K36me3 and H3K27ac, thought to mark active enhancers and transcribed gene bodies, associate with higher expression and partly explain the higher activity of Weak Enhancers over Enhancer predictions. While DNase I hypersensitivity (HS) is a good predictor of active sequences in our assay, transcription factor (TF) binding models need to be included in order to accurately identify highly expressed sequences. Overall, our results show that a significant fraction (∼26%) of the ENCODE enhancer predictions have regulatory activity, suggesting that histone modification states can reflect the cis-regulatory activity of sequences in the genome, but that specific sequence preferences, such as TF-binding sites, are the causal determinants of cis-regulatory activity.
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40.5% of ATAC peaks are active by MPRA, consistent with other studies.
“In other words, 40.5% of ATAC peaks are active by MPRA, consistent with other studies37.”
From Single-cell spatial multi-omics and deep learning dissect enhancer-driven gene regulatory networks in liver zonation (2024) · cites this paper as [37]
The Discussion (page unavailable) reports that only about 26% of predicted enhancer sequences were active in its reporter assay, which supports the broad comparison that not all tested candidate regions are active. The paper does not report ATAC peaks or a 40.5% activity rate, so it cannot substantiate that specific figure or population. (LLM, conf=0.84)
Experimental — accuracy not yet measuredIn mammals, the ratio of accessible regions that are active enhancers drops to approximately 30%.
“but in mammals this ratio drops to ~30%13.”
From Enhancer grammar of liver cell types and hepatocyte zonation states (2022) · cites this paper as [13]
The Discussion (page unavailable) reports that approximately 26% of predicted enhancer sequences were active in the assay, close to the cited ~30%. But the denominator was ENCODE-predicted enhancers tested in a human K562 cell-line assay, not all accessible regions in mammals; the paper also calls its estimate a lower bound. (LLM, conf=0.87)
Experimental — accuracy not yet measuredThe correlation between functional activity measured at scale (e.g. via MPRAs) and the annotations typically used to call enhancers is modest at best.
“Furthermore, to the extent that functional activity has been measured at scale, e.g. via MPRAs, its correlation with the annotations typically used to call enhancers is modest at best106-108.”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [107]
40.5% of ATAC peaks are active by MPRA.
“40.5% of ATAC peaks are active by MPRA, in line with other studies13.”
From Enhancer grammar of liver cell types and hepatocyte zonation states (2022) · cites this paper as [13]
The Results section (“Sequence and chromatin features”; page unavailable) relates DNA accessibility to activity in a reporter assay, but it discusses DNase I hypersensitivity rather than ATAC peaks. Searches for ATAC peaks and 40.5% found no passage reporting the claimed proportion. (LLM, conf=0.76)
Experimental — accuracy not yet measured
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Percentages, sums and reported differences re-computed from the paper’s own numbers.
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- Authors
- Kwasnieski, J. C., Fiore, C., Chaudhari, H. G., & Cohen, B. A.
- Affiliations
- Washington University in St. Louis (US)
- Corresponding author
- Cohen, Barak A.
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2014-07-17
- Volume
- 24
- Issue
- 10
- Pages
- 1595-1602
- ISSN
- 1088-9051 (ISSN-L)
- 1549-5469
- PMC
- PMC4199366
- OpenAlex
- W2007694108
- Cited by
- 224
- References
- 2
- Citations by year
- 2026: 3 · 2025: 15 · 2024: 7 · 2023: 16 · 2022: 24 · 2021: 20 · 2020: 30 · 2019: 19 · 2018: 24 · 2017: 23 · 2016: 24 · 2015: 17 · 2014: 2
- Type
- posted content
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by-nc
- Locations
- Genome Research (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Digital Commons@Becker (Washington University School of Medicine) (submittedVersion, cc-by-nc)
- HighWire Press Open Archive (submittedVersion)
- Funders
- National Institutes of Health (R01 GM092910)
- 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
- ENCODE, histone modifications, enhancers, K562 cells, H3K36me3, H3K27ac, DNase I hypersensitivity, transcription factor binding sites, high-throughput functional assay
- MeSH terms
- Gene Expression Regulation, Histones, Humans, Models, Genetic, Regulatory Sequences, Nucleic Acid, Logistic Models, Sequence Analysis, RNA, Computational Biology, K562 Cells, Embryonic Stem Cells
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