The Structure-Selective Endonucleases GEN1 and MUS81 are Functionally Complementary in Safeguarding the Genome of Proliferating B Lymphocytes

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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. Recombination-dependent DNA transactions that preserve the integrity of the genome and of the DNA replication process generates Holliday junctions that must be eliminated for the accurate segregation of sister chromatids and faithful propagation of genomic material. To investigate the role of two Holliday junction resolvases, 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 halts their development and maturation while selective loss of the resolvases 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 resolvase-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 essential roles of GEN1 and MUS81 in safeguarding the genome to ensure the proper development and maintenance of B lymphocytes.

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  1. Evaluation Summary:

    This manuscript is of interest to individuals working on genome stability and B lymphocyte development. Using knockouts for the genes encoding the structure-selective endonucleases GEN1 and MUS81 in mice, the authors show that the absence of both proteins is incompatible with embryonic development, with selective loss in mature B-cells inhibiting germinal center formation. This is the first study of these enzymes in an organismic context and in primary cells, revealing insight into the in vivo consequences of loss of MUS81 and GEN1 functions not previously accessible through studies in cultured cells.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    The authors generated a mouse model for GEN1 and a B-cell conditional MUS81 flox allele to create B-cells devoid of both proteins using two Cre drivers. Mb1-Cre driven by the Cd79a promoter depletes MUS81 in pre-pro B cells, whereas Cd23-Cre is driven by the Fcer2a promotor to deplete MUS81 in immature, transitional and late B cells. The results show that GEN1 and MUS81 are necessary for differentiation into pro-B cells, the expansion and maintenance of pro-B cells, or both. The CD23-Cre line showed that naïve B cells can persist without MUS81 and GEN1, but they are impaired for germinal center formation. Ex vivo analysis identified a proliferation defect upon stimulation and increased cell death due to apoptosis. RNA seq analysis of such cells revealed activation of the p53 pathway and the type I interferon response. Metaphase chromosomal analysis revealed hallmarks of genomic instability including chromosomal abnormalities, including breaks, fragments, fusions, and radials. The symmetry of the breakages suggests that they arose from unresolved HR intermediates.

    This is a well-executed study, and the results support the conclusions. While the role of both nucleases has been extensively studied in cellular systems, this is the first analysis at the organismic level with a focus on B-cell development.

    The nature of the actual substrate(s) for MUS81 and GEN1 remains unclear and the significance of unresolved HR intermediates for the observed phenotype remains an inference from the type of chromosome aberrations observed. The DNA damage analyses presented would benefit from being clarified and extended.

  3. Reviewer #2 (Public Review):

    In this study, Fernandez et al. investigated the role of the structure-selective endonucleases MUS81 and GEN1 in B cell development and survival. Using conditional deletion of MUS81 in combination with a GEN1 germline knockout strain, the authors show that selective loss of both resolvases in early B cell precursors results in a developmental block of B lymphocytes at the stage of pro-B cells, while deletion of the resolvases in mature naïve B cells impairs both spontaneous and induced germinal center formation. These phenotypes are accompanied by cell cycle arrest, accumulation of cells in the G2/M phase of the cell cycle, increased cell death, and high levels of chromosomal aberrations. Taken together, these observations underscore the role of MUS81 and GEN1 in preserving genome integrity during proliferative blasts occurring during B lymphocyte development and activation.

    Overall, the results presented in this study are robust, thoroughly controlled, and the well-executed work is appreciated. Furthermore, given the relevance of the findings for B cell physiology and genome integrity, this study is in principle of interest to a broad readership. However, a major concern is that this line of work has been already described in other cell types (e.g. Chan Nat. Cell Biol, 2018, Wechsler Nature, 2011), and the observed phenotypes are to some extent expected based on the high proliferative nature of B cells at specific developmental stages and the reported roles of the two resolvases.

    Nonetheless, there are interesting observations arising from the results, which could potentially provide additional and unexpected findings from the B cell model of double-resolvase ablation.

  4. Reviewer #3 (Public Review):

    To investigate their role in B cell development and function, the authors conditionally delete of the structure-specific endonucleases GEN1 and MUS81 at early and late stages of B cell development. Using MB1-Cre, the authors find GEN1 and MUS81 play redundant and essential roles in B cell development, leading to an almost complete depletion of B cells in the pro-B and later stages that was rigorously shown. Conditional deletion of Mus81 in transitional B cells by CD23-cre circumvented this developmental delay, but led to a severe defect in germinal center formation in lymph nodes, Peyer's patches and the spleen specifically in double-deficient cells though total B cell numbers were similar to WT. Further characterization by in vitro stimulated cells revealed that loss of both Gen1 and Mus81 dramatically reduces cell proliferation, induces G2/M checkpoint activation, apoptosis and genome instability. The authors conclude that these defects are caused by MUS81/GEN1's shared role in processing recombination intermediates created by replication stress but do not show the cells experience replication stress. Further, there is no characterization of class switch recombination or IgH damage in the cells, which feels noticeably absent. Finally, the DNA damage analyses presented would benefit from being clarified and extended.

    Overall this is an elegant and straightforward dissection of the role of GEN1 and MUS81 in B cell development, but in its current form the manuscript does not directly connect the observed phenotypes to the molecular role of GEN1/MUS81 in DSB repair.