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).
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
-
- 1,279
- views
-
- 209
- downloads
-
- 2
- 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)
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
Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here, we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Although gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.
-
- Chromosomes and Gene Expression
O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a ‘decoy exon’ present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.
