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.
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mRNA sequencing of control and resolvase-deficient ex vivo-stimulated B cellsNCBI Gene Expression Omnibus, GSE195734.
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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.
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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
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Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
