Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements
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Abstract
Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. However, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that together activate Fgf5 expression during exit from naive murine pluripotency. Four intergenic elements form a super-enhancer, and most of the elements contribute to Fgf5 induction at distinct time points. A fifth, poised enhancer located in the first intron contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. Despite low individual enhancer activity, together these elements strongly induce Fgf5 expression in a super-additive fashion that involves strong accumulation of RNA polymerase II at the intronic enhancer. Finally, we observe a strong anti-correlation between RNA polymerase II levels at enhancers and their distance to the closest promoter, and we identify candidate elements with properties similar to the intronic enhancer.
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Facilitators, as described here, may be relatively common elements in multipartite enhancers.
“Review of diverse, albeit incomplete, previous analyses of SEs16,17,19,20,22 suggest that facilitators, as described here, may be relatively common elements in multipartite enhancers.”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [22]
The Abstract (p. 1) identifies an amplifier element in the Fgf5 enhancer cluster, and the Discussion (p. 16) anticipates that studies of other loci may find additional such elements. The paper does not establish or suggest that these elements are relatively common in multipartite enhancers. (LLM, conf=0.83)
Experimental — accuracy not yet measuredSome deviations from enhancer additivity could potentially be explained by steric constraints arising from enhancer competition or involvement of pioneer enhancers, but these considerations may not readily explain other reported cases.
“Some deviations from enhancer additivity could potentially be explained by steric constraints arising from enhancer competition [519] or involvement of “pioneer enhancers” [525], but these considerations may not readily explain other reported cases [523, 526].”
From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [523]
The Discussion (pp. 14, 16) reports super-additive enhancer action and proposes that PE amplifies the other elements’ contributions, supporting an enhancer-based explanation for a deviation from additivity. But the paper calls PE a “poised enhancer” (p. 4), not a “pioneer enhancer,” and does not identify pioneer enhancers as the explanation. The steric-competition and other-cases clauses are attached to different citations. (LLM, conf=0.82)
Experimental — accuracy not yet measuredA number of groups have dissected super-enhancers, yielding various conclusions, from additive to super-additive, redundant, synergistic, and hierarchical cooperation.
“A number of groups have dissected SEs, yielding various conclusions, from additive16 to super-additive,22 redundant,19 synergistic,21 and hierarchical20 cooperation.”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [22]
Synergistic effects were observed in mammalian cell fate determination systems, for example, at Fgf5 enhancers during differentiation of mouse embryonic stem cells to epiblast-like cells and at up to 20% enhancers upon transdifferentiation of human leukemia B-cells into macrophages.
“synergistic effects were observed in mammalian cell fate determination systems, for example, at Fgf5 enhancers during the differentiation of mouse embryonic stem cells to epiblast-like cells (EpiLCs) [523] and at up to 20% enhancers upon transdifferentiation of human leukemia B-cells into macrophages [524].”
From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [523]
Current theories propose liquid-liquid phase separation and some form of transcription factor trapping or engagement of transcription factors in multivalent interactions independent of assembly into phase-separated liquid-like droplets.
“current theories propose liquid-liquid phase separation18,64–66 and some form of TF trapping22,67 or engagement of TFs in multivalent interactions independent of assembly into phase-separated liquid-like droplets.68”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [22]
The citation is attached to the proposed engagement of transcription factors in multivalent interactions without liquid-like droplets. The Discussion (p. 13) mentions phase separation as one proposed mechanism, while p. 15 speculates that a combination of transcription factors and co-activators could contribute to Pol II accumulation. Neither passage addresses multivalent transcription-factor interactions independent of droplet assembly (searched those terms and a paraphrase). (LLM, conf=0.73)
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Cites
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- Authors
- Thomas, H. F., Kotova, E., Jayaram, S., Pilz, A., Romeike, M., Lackner, A., Penz, T., Bock, C., Leeb, M., Halbritter, F., Wysocka, J., & Buecker, C.
- Affiliations
- University of Vienna (AT)
- Austrian Academy of Sciences (AT)
- St. Anna Children's Cancer Research Institute (AT)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Buecker, Christa
- ORCID
- Venue
- Molecular Cell
- Publisher
- Elsevier BV
- Published
- 2021-03
- Volume
- 81
- Issue
- 5
- Pages
- 969-982.e13
- ISSN
- 1097-2765 (ISSN-L)
- OpenAlex
- W3122541093
- Cited by
- 98
- References
- 12
- Citations by year
- 2026: 17 · 2025: 18 · 2024: 18 · 2023: 17 · 2022: 12 · 2021: 16
- FWCI
- 5.10
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (vor, 368 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Molecular Cell (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Funders
- Universität Wien
- Austrian Science Fund (P 30599)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Keywords
- enhancer clusters, super-enhancers, FGF5, intronic enhancer, RNA polymerase II, transcriptional activation, enhancer function, gene expression regulation, mouse embryo
- MeSH terms
- Mouse Embryonic Stem Cells, Red Fluorescent Protein, Animals, Cell Line, Cell Nucleus, Drosophila melanogaster, Enhancer Elements, Genetic, Exons, Histones, Introns, Luminescent Proteins, Promoter Regions, Genetic, RNA Polymerase II, Transcription, Genetic, Signal Transduction, Sequence Analysis, RNA, Genes, Reporter, Gene Expression Regulation, Developmental, Green Fluorescent Proteins, Mice, Fibroblast Growth Factor 5, Gene Knockout Techniques, Single-Cell Analysis
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