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Activity-based CRISPR Scanning Uncovers Allostery in DNA Methylation Maintenance Machinery
Open accessPreprint — published as 10.7554/elife.80640
Abstract
Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite their fundamental importance, systematic identification of allosteric sites remains highly challenging. Here we perform CRISPR scanning on the essential maintenance methylation machinery—DNMT1 and its partner UHRF1—with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function mutations that increase DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD–PBR interface. Collectively, our findings highlight the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, even beyond the direct drug target.
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Metadata
- Authors
- Ngan, K. C., Hoenig, S. M., Gosavi, P. M., Tanner, D. A., Lue, N. Z., Garcia, E. M., Lee, C., & Liau, B. B.
- Affiliations
- Broad Institute (US)
- Harvard University (US)
- Corresponding author
- Liau, Brian B.
- ORCID
- Venue
- eLife
- Publisher
- openRxiv
- Published
- 2022-05-15
- Volume
- 12
- ISSN
- 2050-084X (ISSN-L)
- PMC
- PMC9946446
- OpenAlex
- W4319825952
- Cited by
- 16
- References
- 1
- Citations by year
- 2026: 3 · 2025: 5 · 2024: 3 · 2023: 4 · 2022: 1
- FWCI
- 1.34
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- eLife (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Science Foundation (1745303)
- Damon Runyon Cancer Research Foundation (1DP2GM137494)
- Harvard University (DGE1745303)
- Health Resources in Action
- National Science Foundation of Sri Lanka (DGE1745303)
- National Institute of General Medical Sciences (DP2GM137494)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- 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
- Keywords
- allosteric regulation, DNA methylation maintenance, DNMT1, UHRF1, decitabine, allosteric site, gain-of-function mutations, mutational hotspots
- MeSH terms
- DNA (Cytosine-5-)-Methyltransferase 1, DNA (Cytosine-5-)-Methyltransferases, DNA Methylation, CCAAT-Enhancer-Binding Proteins, Ubiquitin-Protein Ligases, Clustered Regularly Interspaced Short Palindromic Repeats
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