The structure-selective endonucleases GEN1 and MUS81 mediate complementary functions in safeguarding the genome of proliferating B lymphocytes

Abstract

During the development of humoral immunity, activated B lymphocytes undergo vigorous proliferative, transcriptional, metabolic, and DNA remodeling activities; hence, their genomes are constantly exposed to an onslaught of genotoxic agents and processes. Branched DNA intermediates generated during replication and recombinational repair must be eliminated to preserve the integrity of these DNA transactions for the faithful duplication and propagation of genomic material. To investigate the role of two structure-selective endonucleases, GEN1 and MUS81, in B cell biology, we established B-cell conditional knockout mouse models and found that targeted deletion of GEN1 and MUS81 in early B-cell precursors abrogates the development and maturation of B-lineage cells while selective loss of the enzymes in mature B cells inhibits the generation of robust germinal centers. Upon activation, these double-null mature B lymphocytes fail to proliferate and survive while exhibiting transcriptional signatures of p53 signaling, apoptosis, and type I interferon response. Metaphase spreads of these endonuclease-deficient cells showed severe and diverse chromosomal abnormalities, including a preponderance of chromosome breaks, consistent with a defect in resolving DNA recombination intermediates. These observations underscore the pivotal roles of GEN1 and MUS81 in safeguarding the genome to ensure the proper development and maintenance of B lymphocytes.

Data availability

The RNAseq data and analysis generated in this study are deposited in GEO under the accession code GSE195734. The Gen1 and Mus81 expression data in the various B cell subsets was previously generated by Brazão et al. (2016) and deposited under GSE72018 in GEO.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Keith Conrad Fernandez

    Immunology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Laura Feeney

    Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Ryan M Smolkin

    Immunology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Wei-Feng Yen

    Immunology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Allysia J Matthews

    Immunology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. John HJ Petrini

    Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    For correspondence
    petrinij@mskcc.org
    Competing interests
    The authors declare that no competing interests exist.
  7. Jayanta Chaudhuri

    Immunology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    For correspondence
    chaudhuj@mskcc.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3838-0075

Funding

National Institutes of Health (R01AI072194)

  • Jayanta Chaudhuri

National Institutes of Health (R01AI124186)

  • Jayanta Chaudhuri

National Institutes of Health (R56AI072194)

  • Jayanta Chaudhuri

National Institutes of Health (U54CA137788)

  • Jayanta Chaudhuri

National Institutes of Health (P30CA008748)

  • John HJ Petrini
  • Jayanta Chaudhuri

National Institutes of Health (R01GM56888)

  • John HJ Petrini

National Institutes of Health (R35GM136278)

  • John HJ Petrini

National Institutes of Health (U54OD020355)

  • John HJ Petrini

Geoffrey Beene Cancer Research Center

  • Jayanta Chaudhuri

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

Reviewing Editor

  1. Wolf-Dietrich Heyer, University of California, Davis, United States

Ethics

Animal experimentation: All mice were housed and maintained in groups of five under specific pathogen-free conditions, and euthanized at the time of analyses in accordance with guidelines for animal care established by Memorial Sloan Kettering Cancer Center Research Animal Resource Center and the Institutional Animal Care and Use Committee (IACUC). All mouse experimentation protocols were approved by MSK's IACUC (Protocol Number: 05-12-030).

Version history

  1. Received: January 14, 2022
  2. Preprint posted: February 10, 2022 (view preprint)
  3. Accepted: September 30, 2022
  4. Accepted Manuscript published: October 3, 2022 (version 1)
  5. Version of Record published: October 19, 2022 (version 2)
  6. Version of Record updated: October 26, 2022 (version 3)

Copyright

© 2022, Fernandez 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.

Metrics

  • 988
    views
  • 190
    downloads
  • 1
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Keith Conrad Fernandez
  2. Laura Feeney
  3. Ryan M Smolkin
  4. Wei-Feng Yen
  5. Allysia J Matthews
  6. John HJ Petrini
  7. Jayanta Chaudhuri
(2022)
The structure-selective endonucleases GEN1 and MUS81 mediate complementary functions in safeguarding the genome of proliferating B lymphocytes
eLife 11:e77073.
https://doi.org/10.7554/eLife.77073

Share this article

https://doi.org/10.7554/eLife.77073

Further reading

    1. Chromosomes and Gene Expression
    Rupam Choudhury, Anuroop Venkateswaran Venkatasubramani ... Axel Imhof
    Research Article

    Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity-biotinylation method targeting the RNA and proteins constituents. The method that we termed Antibody-Mediated-Proximity-Labelling-coupled to Mass Spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. This analysis identified a number of known RNA binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.

    1. Cancer Biology
    2. Chromosomes and Gene Expression
    Gregory Caleb Howard, Jing Wang ... William P Tansey
    Research Article

    The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the ‘WIN’ site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.