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LADL: light-activated dynamic looping for endogenous gene expression control
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Abstract
Mammalian genomes are folded into tens of thousands of long-range looping interactions. The cause-and-effect relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short time scales remains an unanswered question. Here, we engineer a new class of synthetic architectural proteins for directed rearrangement of the three-dimensional genome using blue light. We target our light-activated-dynamic-looping (LADL) system to two genomic anchors with CRISPR guide RNAs and induce their spatial colocalization via light-induced heterodimerization of cryptochrome 2 and a dCas9-CIBN fusion protein. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Using single-molecule RNA-FISH, we demonstrate that de novo formation of the Zfp462-SE loop correlates with a modest increase in Zfp462 expression. LADL facilitates colocalization of genomic loci without exogenous chemical cofactors and will enable future efforts to engineer reversible and oscillatory loops on short time scales.
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“Chromatin loops enable interactions between enhancers and promoters enabling fine control of gene expression, in addition to the regulation mediated by transcription factors and other epigenetic modifications”
From Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration (2023) · cites this paper as [98]
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Metadata
- Authors
- Kim, J. H., Rege, M., Valeri, J., Dunagin, M. C., Metzger, A., Titus, K. R., Gilgenast, T. G., Gong, W., Beagan, J. A., Raj, A., & Phillips-Cremins, J. E.
- Affiliations
- University of Pennsylvania (US)
- ORCID
- Venue
- Nature Methods
- Published
- 2019-06-24
- Volume
- 16
- Issue
- 7
- Pages
- 633-639
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC6599567
- OpenAlex
- W2949539957
- Cited by
- 135
- References
- 30
- Citations by year
- 2026: 5 · 2025: 14 · 2024: 12 · 2023: 15 · 2022: 23 · 2021: 33 · 2020: 24 · 2019: 9
- FWCI
- 7.37
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- Nature Methods (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Science Foundation (1562665)
- Foundation for the National Institutes of Health
- Alfred P. Sloan Foundation
- New York Stem Cell Foundation
- National Institutes of Health
- National Heart, Lung, and Blood Institute (U01HL129998)
- National Institute of Mental Health (1DP2MH11024701, DP2MH110247)
- National Institute of General Medical Sciences
- Division of Mathematical Sciences
- Common Fund
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Keywords
- 3D genome organization, CRY2, gene expression regulation, single-molecule RNA FISH
- MeSH terms
- Kruppel-Like Factor 4, RNA, Guide, CRISPR-Cas Systems, Animals, Carrier Proteins, Cells, Cultured, DNA-Binding Proteins, Gene Expression Regulation, Light, Male, Nerve Tissue Proteins, Promoter Regions, Genetic, Protein Engineering, Mice, Kruppel-Like Transcription Factors
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