The structure-selective endonucleases GEN1 and MUS81 mediate complementary functions in safeguarding the genome of proliferating B lymphocytes
- Memorial Sloan Kettering Cancer Center, United States
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.
-
mRNA sequencing of control and resolvase-deficient ex vivo-stimulated B cellsNCBI Gene Expression Omnibus, GSE195734.
-
Long non-coding RNAs in B cells (RNA-Seq)NCBI Gene Expression Omnibus, GSE72018.
Article and author information
Author details
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.
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).
Reviewing Editor
- Wolf-Dietrich Heyer, University of California, Davis, United States
Publication history
- Received: January 14, 2022
- Preprint posted: February 10, 2022 (view preprint)
- Accepted: September 30, 2022
- Accepted Manuscript published: October 3, 2022 (version 1)
- Version of Record published: October 19, 2022 (version 2)
- 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
-
- 646
- Page views
-
- 152
- Downloads
-
- 0
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
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)
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
Further reading
-
- Chromosomes and Gene Expression
- Genetics and Genomics
Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2Δ cells that appears to result directly from impaired decapping rather than elevated transcription. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Edc3 or Scd6; whereas most of the remaining transcripts utilize NMD factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2Δ confers widespread changes in relative translational efficiencies (TEs) that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2&Delta, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are up-regulated, and both mitochondrial function and cell filamentation are elevated in dcp2Δ cells, suggesting that decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.
-
- Chromosomes and Gene Expression
- Genetics and Genomics
Regulatory networks underlying innate immunity continually face selective pressures to adapt to new and evolving pathogens. Transposable elements (TEs) can affect immune gene expression as a source of inducible regulatory elements, but the significance of these elements in facilitating evolutionary diversification of innate immunity remains largely unexplored. Here, we investigated the mouse epigenomic response to type II interferon (IFN) signaling and discovered that elements from a subfamily of B2 SINE (B2_Mm2) contain STAT1 binding sites and function as IFN-inducible enhancers. CRISPR deletion experiments in mouse cells demonstrated that a B2_Mm2 element has been co-opted as an enhancer driving IFN-inducible expression of Dicer1. The rodent-specific B2 SINE family is highly abundant in the mouse genome and elements have been previously characterized to exhibit promoter, insulator, and non-coding RNA activity. Our work establishes a new role for B2 elements as inducible enhancer elements that influence mouse immunity, and exemplifies how lineage-specific TEs can facilitate evolutionary turnover and divergence of innate immune regulatory networks.
