Base editor scanning charts the DNMT3A activity landscape

Lue, Nicholas Z.; García, Emma Moreno; Ngan, Kevin C.; Lee, Ceejay; Doench, John G.; Liau, Brian B.

doi:10.1038/s41589-022-01167-4

Cited by 28 publications

(50 citation statements)

References 60 publications

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“…By coupling a fluorescence-based DNA methylation reporter system with base editing to generate a library of mutants, we previously identified a wide variety of LOF mutations in DNMT3A, and highlighted the importance of the PWWP domain for enzyme activity, particularly in its ability to bind to DNA. 12 However, this study only identified a single activating mutation to DNMT3A, which disrupts the known autoinhibitory ADD-MTase interaction. 10 Whether additional activating mechanisms exist remains an open question.…”

Section: Introductionmentioning

confidence: 82%

“…To identify activating DNMT3A mutations, we sought to adapt our previously reported base editor scanning approach coupled to a fluorescence-based reporter of DNA methylation. 12,17 In this system, we employed a reporter construct that contains TetO sites upstream of a promoter controlling the expression of citrine (Figure 1a). In our original system (v1), we fused DNMT3L, a catalytically inactive accessory protein that binds to DNMT3A 18,19 to rTetR, such that adding doxycycline (dox) to the cells recruited rTetR-DNMT3L to the citrine promoter.…”

Section: Development Of a Screening Approach For Discovery Of Activat...mentioning

confidence: 99%

“…Motivated by these prospects, we conducted in situ mutational scanning on DNMT3A using base editing. 12 Base editing is a form of precision genome editing that has enabled the direct mutational scanning of a variety of endogenous proteins. [13][14][15][16] Base editing has the advantage of targeting the native gene, avoiding the confounding effects of overexpressing mutants of a protein of interest.…”

Section: Introductionmentioning

confidence: 99%

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Base Editor Scanning Reveals Activating Mutations of DNMT3A

García

Lue

Liang

et al. 2023

Preprint

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DNA methyltransferase 3A (DNMT3A) is ade novocytosine methyltransferase responsible for establishing proper DNA methylation during mammalian development. Loss-of-function (LOF) mutations to DNMT3A, including the hotspot mutation R882H, frequently occur in developmental growth disorders and hematological diseases, including clonal hematopoiesis (CH) and acute myeloid leukemia (AML). Accordingly, identifying mechanisms that activate DNMT3A is of both fundamental and therapeutic interest. Here, we applied a base editor mutational scanning strategy with an improved DNA methylation reporter to systematically identify DNMT3A activating mutations in cells. By integrating an optimized cellular recruitment strategy with paired isogenic cell lines with or without the LOF hotspot R882H mutation, we identify and validate three distinct hyperactivating mutations within or interacting with the regulatory ADD domain of DNMT3A, nominating these regions as potential functional target sites for pharmacological intervention. Notably, these mutations are still activating in the context of a heterozygous R882H mutation. Altogether, we showcase the utility of base editor scanning for discovering functional regions of target proteins.SynopsisUsing base editor mutagenesis and a DNA methylation reporter optimized to find activating mutations, we identify novel hyperactivating mutations in DNMT3A that suggest new mechanisms of allosteric control.

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Section: Introductionmentioning

confidence: 82%

Section: Development Of a Screening Approach For Discovery Of Activat...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Base Editor Scanning Reveals Activating Mutations of DNMT3A

García

Lue

Liang

et al. 2023

Preprint

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“…However, smaller, more precise perturbations such as point mutations may be more appropriate for other biological targets. In such cases, our activity-based approach, which is primarily focused on the selection modality (i.e., mechanistic inhibitors), can also accommodate alternative editing technologies such as base editing or prime editing ( Anzalone et al, 2019 ; Anzalone et al, 2020 ; Hanna et al, 2021 ; Lue et al, 2023 ) to assess distinct mutational perturbations. Indeed, we envision that integration of diverse editing modalities will further increase the utility of our activity-based approach.…”

Section: Discussionmentioning

confidence: 99%

Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery

Ngan

Hoenig

Kwok

et al. 2023

eLife

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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.

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“…For instance, DNMT1 mutations have been described in colorectal cancer [ 29 ], and the disrupted activity of DNMT1 in mice was reported to induce repetitive insertions of a transposable element within the Notch gene leading to its oncogenic activation in thymic lymphomas [ 26 ]. In addition, loss-of-function mutations in the DNMT3A gene are one of the most frequent defects identified in acute myeloid leukemia (AML; ~30%) or myelodysplastic syndrome (MDS; 2–10%) patients [ 30 ]. Intriguingly, over half of DNMT3A mutations occur at the R882 amino acid residue in AML patients.…”

Section: Dna Methyltransferases and Their Inhibitorsmentioning

confidence: 99%

Epigenetic Regulators of DNA Cytosine Modification: Promising Targets for Cancer Therapy

Jung

2023

Biomedicines

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Epigenetic modifications are crucial regulators of gene expression that critically impact cell lineage differentiation, survival, and proliferation, and dysregulations are commonly observed in various cancers. The aberrantly modified epigenome confers unique features on tumor cells, including sustained proliferative potential, resistance to growth-suppressive or cell death signals, augmented replicative immortality, invasion, and metastasis. As a result, epigenetic abnormalities exhibit significant impacts on all stages of oncogenesis from its onset to progression to metastasis. Among various epigenetic mechanisms in mammals, DNA cytosine methylation–demethylation is recurrently disrupted in cancers. Due to its inherent reversibility, targeting DNA methylation dynamics has gained tremendous attention as a promising therapeutic option that can ameliorate the effects of cancer-specific epigenetic abnormalities by restoring normal conditions. Various small molecules targeting DNA (de)methylation regulators have been developed as potential cancer therapeutics, some of which are approved for usage in clinics. Clinical trials of many other molecules are underway for both hematological malignancies and solid tumors. In this review, we discuss the DNA methylation/demethylation pathway as a promising target for therapeutic intervention in cancer and highlight the development of various epigenetic drugs targeting DNA-modifying enzymes such as DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) enzymes.

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Base editor scanning charts the DNMT3A activity landscape

Cited by 28 publications

References 60 publications

Base Editor Scanning Reveals Activating Mutations of DNMT3A

Base Editor Scanning Reveals Activating Mutations of DNMT3A

Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery

Epigenetic Regulators of DNA Cytosine Modification: Promising Targets for Cancer Therapy

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