FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis - PubMed (original) (raw)

. 2015 Sep 15;24(18):5234-49.

doi: 10.1093/hmg/ddv244. Epub 2015 Jun 29.

Affiliations

FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis

Yasuko Kato et al. Hum Mol Genet. 2015.

Abstract

Fanconi anemia (FA) is a recessive X-linked and autosomal genetic disease associated with bone marrow failure and increased cancer, as well as severe germline defects such as hypogonadism and germ cell depletion. Although deficiencies in FA factors are commonly associated with germ cell defects, it remains unknown whether the FA pathway is involved in unique epigenetic events in germ cells. In this study, we generated Fancb mutant mice, the first mouse model of X-linked FA, and identified a novel function of the FA pathway in epigenetic regulation during mammalian gametogenesis. Fancb mutant mice were infertile and exhibited primordial germ cell (PGC) defects during embryogenesis. Further, Fancb mutation resulted in the reduction of undifferentiated spermatogonia in spermatogenesis, suggesting that FANCB regulates the maintenance of undifferentiated spermatogonia. Additionally, based on functional studies, we dissected the pathway in which FANCB functions during meiosis. The localization of FANCB on sex chromosomes is dependent on MDC1, a binding partner of H2AX phosphorylated at serine 139 (γH2AX), which initiates chromosome-wide silencing. Also, FANCB is required for FANCD2 localization during meiosis, suggesting that the role of FANCB in the activation of the FA pathway is common to both meiosis and somatic DNA damage responses. H3K9me2, a silent epigenetic mark, was decreased on sex chromosomes, whereas H3K9me3 was increased on sex chromosomes in Fancb mutant spermatocytes. Taken together, these results indicate that FANCB functions at critical stages of germ cell development and reveal a novel function of the FA pathway in the regulation of H3K9 methylation in the germline.

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Figures

Figure 1.

Figure 1.

FANCB localization on meiotic sex chromosomes and generation of Fancb mutant mice. (A) Immunostaining of meiotic chromosome spreads using an anti-mouse FANCB antibody. SYCP3 is a marker for meiotic chromosome axes. Dotted squares represent the areas surrounding sex chromosomes, and the areas are magnified in the right panels. Scale bars: 10 µm. (B) Structure of Fancb gene and FANCB protein. The site of ZFN-induced mutation of line #84 mutant mice is shown. Asterisk indicates targeting site. White boxes represent non-coding regions, and gray boxes represent coding regions. The mutation leads to a frame change (shown with a black box) and a premature stop codon. (C) Immunoprecipitation and western blot analysis showing the absence of full-length FANCB protein in Fancb mutant testes. (D) Body weights compared for wild-type and Fancb mutant mice at 4 and 10 weeks after birth. Mean and SEM for independent mice are shown. Numbers of mice analyzed at 4 and 10 weeks, respectively: seven and six wild-type, and seven and six Fancb mutant mice. *P < 0.05. Unpaired _t_-test. (E) Fertility test: mean numbers of litters and SEM for independent mice are shown. Numbers of mice tested: seven wild-type and seven Fancb mutant mice. P < 0.05. Unpaired _t_-test. (F) Testis weights/body weight (mg/g) of wild-type and Fancb mutant mice at 4 and 10 weeks. Mean and SEM for independent mice are shown. Numbers of mice analyzed at 4 and 10 weeks, respectively: seven and six wild-type, and seven and six Fancb mutant mice. *P < 0.05. Unpaired _t_-test. (G) Representative photo of the testes of wild-type and Fancb mutant mice. (H) Immunostaining of testicular sections for H4K16 acetylation and protamine 1 (PRM1) from 10-week-old wild-type and Fancb mutant mice. Scale bars: 100 µm. The areas shown with white dotted squares are magnified in the right panels.

Figure 2.

Figure 2.

Reduction of PGCs in Fancb mutant mice. (A–C) Immunostaining of mouse whole embryos at E9.5, E11.5 and E13.5. SOX2 is a marker for PGCs, and GATA4 is a marker for gonadal somatic cells. Scale bars: 100 µm. (D) Total numbers of the SOX2-positive PGCs per embryo at E9.5. (E) Total numbers of PGCs in each genital ridge (cells/genital ridge) at E11.5. Mean and SEM for independent mice are shown. Numbers of mice analyzed at E9.5 and E11.5, respectively: five wild-type and four Fancb mutant mice. *P < 0.05. Unpaired _t_-test.

Figure 3.

Figure 3.

FANCB regulates the maintenance of undifferentiated spermatogonia. (A) Immunostaining of testicular sections at 10 weeks old. WT1 is a marker for Sertoli cells. Dotted circles are the areas of representative tubules. (B) Quantification of seminiferous tubule areas (103 μm2) at 4 and 10 weeks after birth. Mean and SEM for independent mice are shown. A total of 100 tubules from three independent mice were analyzed at 4 and 10 weeks, for both wild-type and Fancb mutant mice. (C) TUNEL assays of testicular sections at 10 weeks old. (D) Scoring of the number of TUNEL-positive cells per germ cell-containing tubule. Mean and SEM for independent mice are shown. Total numbers of tubules analyzed at 4 and 10 weeks, respectively: 303 and 430 wild-type, and 394 and 138 Fancb mutant mice. Three independent mice were analyzed for each dataset. (E and F) Immunostaining of testicular sections at 10 weeks old for panels in E and at 6-months old for panels in F. PLZF is a marker for undifferentiated spermatogonia. Arrowheads: PLZF-positive cells. (G) Scoring of the percentage of PLZF-positive tubules per total tubule. One hundred tubules from three independent mice were analyzed at 4 weeks, 10 weeks and 6 months, in both wild-type and Fancb mutant mice. Mean and SEM for independent mice are shown. (H) Scoring of the number of PLZF-positive cells per PLZF-positive tubule. One hundred tubules from three independent mice were analyzed at 4 weeks, 10 weeks and 6 months, both for wild-type and Fancb mutant mice. Mean and SEM for independent mice are shown. (I and J) Immunostaining of testicular sections at 10 weeks old for panels in I, and at 6 months old for panels in J. (K) Scoring of the percentage of γH2AX-positive tubules per total tubule. Mean and SEM for independent mice are shown. One hundred tubules from three independent mice were analyzed at 4 and 10 weeks, in both wild-type and Fancb mutant mice. One hundred tubules (two independent mice) were analyzed at 6 months, in both wild-type and Fancb mutant mice. Scale bars: 100 µm. *P < 0.05. Unpaired _t_-test.

Figure 4.

Figure 4.

Roles of FANCB in meiotic recombination. (A) Schematic of meiotic prophase and the involvement of two DDR events. (B) The percentage of each stage of meiotic prophase as judged by SYCP3 immunostaining. Total numbers of cells analyzed: 263 from wild-type, and 230 from Fancb mutant mice. Mean and SEM for independent mice are shown. Three independent mice were analyzed for each dataset. (C–I) Immunostaining of chromosome spreads. Scale bars: 10 µm. (C–E) RAD51 foci in zygotene and pachytene spermatocytes. In the right panels: scoring of the number of RAD51 foci per cell at the indicated stages. Distribution of data is shown as dots; mean ± SEM for independent mice is shown as bars. Total numbers of analyzed nuclei are indicated in the panels. For each panel, four independent mice were analyzed for both wild-type and Fancb mutant mice. (F) SYCP1 localization in pachytene spermatocytes. (G and H) FANCD2 foci in zygotene and pachytene spermatocytes. (I) MLH1 foci in pachytene spermatocytes. In the right panel: scoring of the number of MLH1 foci per cell at the mid pachytene stage. Distribution of data is shown as dots; mean ± SEM for independent mice is shown as bars. Total numbers of analyzed nuclei are indicated in the panel. Five independent mice were analyzed for both wild-type and Fancb mutant mice. Arrows: sex chromosomes; arrowheads: RAD51 foci in panel (E) and FANCD2 foci in panel (H). *P < 0.05. ns: not significant. Unpaired _t_-test.

Figure 5.

Figure 5.

Regulation of DDR factors on the sex chromosomes in Fancb mutant mice. (A–G and I) Immunostaining of chromosome spreads of pachytene spermatocytes. In panel G, dotted squares represent areas surrounding sex chromosomes, and the areas are magnified in the bottom panels. Arrows: sex chromosomes. Scale bars: 10 µm unless otherwise indicated. (H) Scoring of the number of RAD51 foci per X axis, Y axis, or pseudo-autosomal region (PAR) in mid-pachytene spermatocytes. Distribution of data is shown as dots; mean ± SEM for independent mice is shown as bars. Total numbers of analyzed nuclei are indicated in the panel. Four independent mice were analyzed for both wild-type and Fancb mutant mice. *P < 0.05. Unpaired _t_-test. (J) Model of the role of FANCB in DDR pathways on the sex chromosomes during meiosis.

Figure 6.

Figure 6.

MDC1 is required for FANCB localization on the sex chromosomes. (A–F) Immunostaining of chromosome spreads of pachytene spermatocytes from mice that are mutant for different DDR factors as indicated. Dotted squares represent areas surrounding sex chromosomes, and the areas are magnified in the right panels. Scale bars: 10 µm unless otherwise indicated.

Figure 7.

Figure 7.

FANCB regulates H3K9 methylation on sex chromosomes. (A–F, H, I, K and L) Immunostaining of chromosome spreads. Arrows: sex chromosomes. Scale bars: 10 µm. (G and J) Quantification of H3K9me2/3 signal intensity on sex chromosomes and autosome regions. Relative Mean Fluorescence Intensity (RMFI) was calculated as described in the Materials and Methods section. Mean and SEM for independent mice are shown. Numbers of analyzed nuclei are indicated in the panels. Three independent mice were analyzed for both wild-type and Fancb mutant mice. *P < 0.05 by one-way ANOVA followed by Tukey's range test.

Figure 8.

Figure 8.

Model of a FANCB-regulated epigenetic pathway on the sex chromosomes. Summary of the FANCB-regulated pathway, and SCML2- and RNF8-dependent pathways, on the sex chromosomes during meiosis. The SCML2- and RNF8-dependent pathways were identified previously.

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