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

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. FancjGT/GT mutants are fertile, but their testes are very much smaller than wild-type litter-mates, 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 FancjGT/GT males, and the FANCJ-interacting protein BRCA1 assembles normally on meiotic chromosome cores. However, MLH1 focus frequency is increased in FancjGT/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 FancjGT/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.

Cohesin Removal along the Chromosome Arms during the First Meiotic Division Depends on a NEK1-PP1γ-WAPL Axis in the Mouse

“…Prophase I spermatocyte chromosome spreads were performed as previously described (Sun et al, 2015). Primary antibodies included: rabbit anti-Sororin (anti-Sororin serum C-106 was generated using a full-length recombinant mouse protein cloned in pET-12a vector and expressed in E. coli (Carretero et al, 2013)), anti-mouse SYCP3 (ab97672), rabbit anti-PDS5B (ab84918) (both from Abcam.…”

Dynamic transcriptome profiles within spermatogonial and spermatocyte populations during postnatal testis maturation revealed by single-cell sequencing

“…Our data presented here demonstrate age-related upregulation of pathways associated with DNA replication and repair, double strand break repair, and response to DNA damage, all of which may underlie the well-characterized differences between spermatocytes in the first wave compared to steady-state spermatocytes. Included in these sets of variably-expressed genes are known regulators of DNA damage response and cross-over formation including Rad51 33 , Brip1 29 , and H2afx 34 , as well as Brca1 and Brca2 30–32 and Atm 35,59 , all of which increase in spermatocytes with age, effects which we have also shown at the protein level for both RAD51 and ATM ( Figures 10, S11, S12 ). While the cause of this lower expression in the first wave is unknown, it has been demonstrated that spermatocytes from juvenile mice generate only about 25% of the double strand breaks present in spermatocytes from steady-state spermatogenesis 59 .…”

“…From this analysis, we observe decreasing expression of genes related to translation and post-transcriptional regulation, and increasing expression of genes related to DNA replication, double strand break repair, and cell cycle regulation ( Figure 7 & S6B, Table S6 ). Most notable in the list of genes upregulated in spermatocytes of increasing age are those known to be essential to DNA repair, meiotic progression, and crossover formation including Brip1 29 , Brca1 and Brca2 30–32 , Rad51 33 , H2afx 34 and Atm 35 . Many of these pathways, particularly those related to double strand break repair (which initiates meiotic recombination), may be crucial for understanding the molecular mechanisms underlying fundamental differences in first-wave spermatocytes.…”

Role of HP1β during spermatogenesis and DNA replication

“…During the germ cell reproductive process, epigenetic chromatin modifications such as methylation, acetylation and ubiquitination play major roles (Gannon et al, 2014). (Guo et al, 2018;Simhadri et al, 2014;Sun et al, 2016;Ward et al, 2003).…”

“…In dividing cells HR predominantly occurs in mitotic S phase cells to maintain DNA fidelity During meiosis phase 1 the many double strand breaks generated during crossover are also repaired by HR (Cohen and Pollard, 2001;Roeder, 1997). Loss of HR repair related proteins, therefore, (such as 53BP1, RIF1, BRCA1, ATM, MOF) affects spermatogenesis (Bartkova et al, 2001;Jiang et al, 2018;Marcet-Ortega et al, 2017;Pandita et al, 1999;Schwab et al, 2013;Sun et al, 2016). Cbx1 -/mice display prenatal death due to severe alveoli lung damage and abnormal neuromuscular and cerebral cortex tissue development (Aucott et al, 2008).…”