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. 2020 Jun;159(6):659-668.
doi: 10.1530/REP-19-0436.

FANCD2 is required for the repression of germline transposable elements

Yan Nie  1 , Andrew F Wilson  1 , Tony DeFalco  2   3 , Amom Ruhikanta Meetei  1   3 , Satoshi H Namekawa  2   3 , Qishen Pang  1   3
Affiliations

FANCD2 is required for the repression of germline transposable elements

Yan Nie et al. Reproduction. 2020 Jun.

Abstract

The Fanconi anemia (FA) DNA damage response (DDR) pathway regulate important cellular processes such as DNA replication, cell cycle control and DNA damage repair. Here we show that FANCD2, a key member of the FA DDR pathway, interacts with several important components of the germ-cell-specific Prmt5/piRNA pathways that orchestrate the repression of transposable elements (TEs). By using the Pou5f1-eGFP reporter mice, which marks pure populations of primordial germ cells (PGCs), we demonstrate that FA deficiency results in de-repression of TEs, depletion of PGCs, and defective spermatogenesis and oogenesis. Fancd2–KO PGCs exhibited excessive DNA damage and exacerbated apoptosis. Mechanistically, we observed a significant reduction of PRMT5-catalyzed H2A/H4R3me2s marks on the LINE1 TEs in E10.5 PGCs of Fancd2-KO; Pou5f1-eGFP and Fanca-KO;Pou5f1-eGFP embryos. Furthermore, we utilized the Fancd2-KI model to show that Fancd2 and Prmt5 co-occupied the promoter of LINE1 in WT PGCs, and that this co-occupancy was lost in FA-deficient (Fanca-KO) PGCs. These results suggest that the FA pathway takes part in TE repression in early PGCs, likely through a mechanism involving Fancd2-facilitated, Prmt5-catalyzed repressive H2A/H4R3me2s marks on TEs.

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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Fancd2 interacts with germline-specific Prmt5/piRNA factors.
(A) List of tissue specific Fancd2-associated proteins. The testis-specific Prmt5/piRNA factors are marked by bold. (B) Validation of the testis-specific Prmt5/piRNA factors by anti-FLAG immunoprecipitation (IP) from testis lysates and detected by antibodies against the indicated proteins. (C) Western analysis of the indicated Prmt5/piRNA factors in the testes of WT, Fancd2-KI and Fancd2-KO mice.
Figure 2.
Figure 2.. Loss of Fancd2 leads to hypogonadism.
(A) Images of embryos (Left) of the indicated genotypes at E13.5. Genotypes (Right) of the embryos were determined by PCR. (B) Representative photo of the testes and ovary of WT and Fancd2-KO mice. (C) Gonadal defects in Fancd2−/− mice. Shown are H&E stained sections of testes and ovaries from 8-week-old WT and Fancd2−/− mice (200× magnification).
Figure 3.
Figure 3.. Loss of Fancd2 leads to PGC depletion.
(A) Representative dot plots of flow cytometric analysis of GFP+ SSEA1+ PGCs from E11.5 embryos of WT and Fancd2−/− females expressing the Pou5f1-eGFP reporter gene. (B) The number of PGCs at E11.5 and E13.5 embryos of WT and Fancd2−/− females expressing the Pou5f1-eGFP reporter gene was determined by flow cytometry for GFP+ SSEA1+ PGCs. Results are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01
Figure 4.
Figure 4.. Reduced proliferation and increased apoptosis in Fancd2-KO PGCs.
(A) Reduced proliferation in Fancd2-KO PGCs. GFP+ PGCs from E11.5 embryos of WT and Fancd2−/− females expressing the Pou5f1-eGFP reporter gene were sorted by FACS and cultured on STO feeder cells for 15 days. Images were taken at the indicated time points. (B) Cumulative growth curve of PGCs during PGC culture initiation. GFP+ PGCs from E11.5 embryos of WT and Fancd2−/− females expressing the Pou5f1-eGFP reporter gene were sorted by FACS and cultured on STO feeder cells for the indicated days. Values indicate mean ± SD of three independent experiments. *p < 0.05, **p < 0.01. (C) Diminished BrdU incorporation in Fancd2-KO PGCs. GFP+ PGCs from E11.5 embryos of WT and Fancd2−/− females expressing the Pou5f1-eGFP reporter gene were sorted by FACS and subjected to pulse-labeling with BrdU followed by flow cytometric analysis for BrdU-positive cells. Values indicate mean ± SD of three independent experiments. *p < 0.05. (D) Increased apoptosis in Fancd2-KO PGCs. GFP+ PGCs from E11.5 embryos of WT and Fancd2−/− females expressing the Pou5f1-eGFP reporter gene were subjected to flow cytometric analysis for Annexin V-positive, DAPI-negative apoptotic cells. Results are presented as mean ± SD of three independent experiments. **p < 0.01
Figure 5.
Figure 5.. FA deficiency leads to de-repression of germline TEs.
(A) Fancd2 is essential for TE repression in PGCs. qPCR analysis of TEs in FACS-sorted GFP+ SSEA1+ PGCs from E11.5 embryos of WT and Fancd2−/− females expressing the Pou5f1-eGFP reporter gene, using the primer sets described in Materials and Methods. Results are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01. (B) De-repression of germline TEs in Fanca-deficient PGCs. qPCR analysis of TEs in FACS-sorted GFP+ SSEA1+ PGCs from E11.5 embryos of WT and Fanca−/− females expressing the Pou5f1-eGFP reporter gene, using the primer sets described in Materials and Methods. Results are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01. (C) μChIP analysis for H2A/H4R3me2s at the promoter region of LINE1 TEs in FACS-sorted GFP+ SSEA1+ PGCs from E10.5 embryos of WT, Fanca−/− and Fancd2−/− females expressing the Pou5f1-eGFP reporter gene. Percentages of anti- H2A/H4R3me2s immunoprecipitated DNA compared to input are shown. w/o, without primary antibodies. Results are presented as mean ± SD of three independent experiments. *p < 0.05. (D) μChIP analysis for Fancd2 (FLAG) and Prmt5 at the promoter region of LINE1 TEs in FACS-sorted GFP+ SSEA1+ PGCs from E10.5 embryos of WT, Fanca−/− and Fancd2−/− females expressing the Pou5f1-eGFP reporter gene and the Fancd2KI/KI transgene. Percentages of anti-FLAG and anti-Prmt5 immunoprecipitated DNA compared to input are shown. w/o, without primary antibodies. Results are presented as mean ± SD of three independent experiments. *p < 0.05. (E) Western analysis of the Prmt5 and Fancd2 proteins in the in-vitro expanded PGCs from E13.5 WT (WT;Fancd2-KI) and Fanca-deficient (Fanca−/−;Fancd2-KI) embryos, using antibodies against the indicated proteins. (E) Western analysis of the Prmt5 and Fancd2 proteins in the E13.5 PGCs from WT (WT;Fancd2-KI) and Fanca-deficient (Fanca−/−;Fancd2-KI) mice, using antibodies against the indicated proteins. (F) Increased accumulation of DNA damage in Fancd2-deficient PGCs. Genomic DNA was stained with SYBR Gold and imaged after single cell electrophoresis (left). Quantification for the percent of DNA in the comet tail is shown (right). Results are presented as mean ± SD of three independent experiments. *p < 0.05.
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