Abstract

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite the fundamental importance of allosteric sites, their identification 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. In contrast to non-covalent DNMT1 inhibition, activity-based selection implicates numerous regions outside the catalytic domain in DNMT1 function. 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 study highlights the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, and more broadly, introduces new tools and analyses that further refine the CRISPR scanning framework.

Data availability

All data generated or analyzed during this study are included in the manuscript and supplementary files; Source Data for the CRISPR scanning experiments and individual sgRNA validation experiments are provided in Supplementary Files 1-3. The sequences of primers and oligonucleotides used in this study are provided in Supplementary File 4. Custom scripts used to analyze the data are available at https://github.com/liaulab/DNMT1_eLife_2022.

Article and author information

Author details

  1. Kevin Chun-Ho Ngan

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8067-3472
  2. Samuel M Hoenig

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  3. Hui Si Kwok

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  4. Nicholas Z Lue

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4236-9127
  5. Pallavi M Gosavi

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  6. David A Tanner

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  7. Emma M Garcia

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  8. Ceejay Lee

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  9. Brian B Liau

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    For correspondence
    liau@chemistry.harvard.edu
    Competing interests
    Brian B Liau, holds sponsored research projects with Eisai and AstraZeneca, is a scientific consultant for Imago BioSciences and Exo Therapeutics, and is a shareholder and member of the scientific advisory board of Light Horse Therapeutics..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2985-462X

Funding

National Science Foundation (Graduate Research Fellowship,DGE1745303)

  • Kevin Chun-Ho Ngan
  • Nicholas Z Lue
  • Emma M Garcia

National Institute of General Medical Sciences (New Innovator Award,1DP2GM137494)

  • Brian B Liau

Damon Runyon Cancer Research Foundation (Damon Runyon-Rachleff Innovation Award)

  • Brian B Liau

Harvard University (Landry Cancer Biology Fellowship)

  • Emma M Garcia

Harvard University (Herchel Smith Graduate Fellowship)

  • Ceejay Lee

Harvard University (Startup Funding)

  • Brian B Liau

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2023, Ngan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Kevin Chun-Ho Ngan
  2. Samuel M Hoenig
  3. Hui Si Kwok
  4. Nicholas Z Lue
  5. Pallavi M Gosavi
  6. David A Tanner
  7. Emma M Garcia
  8. Ceejay Lee
  9. Brian B Liau
(2023)
Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery
eLife 12:e80640.
https://doi.org/10.7554/eLife.80640

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https://doi.org/10.7554/eLife.80640