Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery
- Harvard University, United States
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
Kevin Chun-Ho Ngan
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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Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery
eLife 12:e80640.
https://doi.org/10.7554/eLife.80640
Further reading
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Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
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