Abstract
Fancj, the gene associated with Fanconi anemia (FA) Complementation Group J, encodes a DNA helicase involved in homologous recombination repair and the cellular response to replication stress. FANCJ functions in part through its interaction with key DNA repair proteins, including MutL homolog-1 (MLH1), Breast Cancer Associated gene-1 (BRCA1), and Bloom syndrome helicase (BLM). All three of these proteins are involved in a variety of events that ensure genome stability, including the events of DNA double strand break (DSB) repair during prophase I of meiosis. Meiotic DSBs are repaired through homologous recombination resulting in non-crossovers (NCO) or crossovers (CO). The frequency and placement of COs are stringently regulated to ensure that each chromosome receives at least one CO event, and that longer chromosomes receive at least one additional CO, thus facilitating the accurate segregation of homologous chromosomes at the first meiotic division. In the present study, we investigated the role of Fancj during prophase I using a gene trap mutant allele. Fancj GT/GT mutants are fertile, but their testes are very much smaller than wild-type littermates, predominantly as a result of impeded spermatogonial proliferation and mildly increased apoptosis during testis development in the fetus. This defect in spermatogonial proliferation is consistent with mutations in other FA genes. During prophase I, early events of synapsis and DSB induction/repair appear mostly normal in Fancj GT/GT males, and the FANCJ-interacting protein BRCA1 assembles normally on meiotic chromosome cores. However, MLH1 focus frequency is increased in Fancj GT/GT males, indicative of increased DSB repair via CO, and is concomitant with increased chiasmata at diakinesis. This increase in COs in the absence of FANCJ is associated with increased localization of BLM helicase protein, indicating that BLM may facilitate the increased rate of crossing over in Fancj GT/GT males. Taken together, these results demonstrate a critical role for FANCJ in spermatogenesis at two stages: firstly in the proliferative activity that gives rise to the full complement of testicular spermatogonia and secondly in the establishment of appropriate CO numbers during prophase I.
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Acknowledgments
We thank Peter Borst for his assistance with mouse handling, genotyping, and maintenance. We are indebted to Dr. Satoshi Namekawa (Cincinnati Children’s Hospital, OH) and Dr. Raimundo Freire (University of Tenerife, Spain) for generously providing antibodies critical for these studies. We thank Simon Boulton (Francis Crick Institute, London) for providing advice and primer sequences for genotyping. We are grateful to Robert Munroe and Christian Abratte of the Cornell Stem Cell and Transgenic Core Facility for performing blastocyst injections. The Cornell Stem Cell and Transgenic Core is supported by NYSDOH contract C024174. This work was supported through funding from the NICHD to J.K.H. (HD065870) and from the NIGMS to P.E.C (GM097263). A.C. was supported by an award from the Dextra Undergraduate Research Endowment fund.
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Laboratory animals were maintained and utilized in accordance with Federal guidelines for the care and use of animals, and under a protocol approved by the Cornell Institutional Animal Care and Use Committee (IACUC). All authors declare that they have no conflicts of interest relating to the work described herein. This work was supported through funding from the NICHD to J.K.H. (HD065870) and from the NIGMS to P.E.C (GM097263). A.C. was supported by an award from the Dextra Undergraduate Research Endowment fund.
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This article is part of a Special Issue on “Recent advances in meiotic chromosome structure, recombination and segregation” edited by Marco Barchi, Paula Cohen and Scott Keeney.
Xianfei Sun, Miguel A. Brieño-Enriquez and Alyssa Cornelius contributed equally to this work.
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Supplementary Figure 1
Protein and gene structure for FANCJ and western blot showing loss of the FANCJ protein in Fancj GT/GT testis. (a) Protein structure of mouse FANCJ showing MLH1 interaction domain, helicase domain, and BRCA1 interaction domain. (b) Gene trap allele showing position of the gene trap cassette after exon 5 of the gene. The cassette contains an FRT-flanked βGeo gene. Genotyping primers are indicated. c Western blot showing the loss of 140 kDa FANCJ protein in testis lysates from homozygous mutant genetrap males. A non-specific band of approximately 96 kDa appears in all samples. The same gel was probed with an antibody against β-Tubulin. (GIF 25 kb)
Figure S2
Growth of wildtype (E14 parental) and Fancj GT/GT ES cells over seven days. At least three ES cell clones were grown for each cell line, beginning with equivalent numbers of cells being seeded onto 35 mm petri dishes. Cells were counted daily and replated in fresh medium as needed. (A) shows percentage of cells over the initial seed number, (B) shows raw cell counts for each clone. (GIF 106 kb)
Supplementary Figure 3
Examples of aberrant spermatocyte chromosome spreads from Fancj GT/GT testis. (a) Three cells stained for SYCP3 (green), the centromere (CREST, magenta) and DNA (DAPI, blue), showing many altered synapsis figures (exemplified by the white arrows). (b,c) Examples of γH2AX staining (red) on the SC (SYCP3, green) during mid-pachynema, showing persistent γH2AX staining. (GIF 49 kb)
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Sun, X., Brieño-Enríquez, M.A., Cornelius, A. et al. FancJ (Brip1) loss-of-function allele results in spermatogonial cell depletion during embryogenesis and altered processing of crossover sites during meiotic prophase I in mice. Chromosoma 125, 237–252 (2016). https://doi.org/10.1007/s00412-015-0549-2
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DOI: https://doi.org/10.1007/s00412-015-0549-2