NRF1 coordinates with DNA methylation to regulate spermatogenesis

Abstract

Spermatogenesis is a highly coordinated process that requires tightly regulated gene expression programmed by transcription factors and epigenetic modifiers. In this study, we found that nuclear respiratory factor (NRF)-1, a key transcription factor for mitochondrial biogenesis, cooperated with DNA methylation to directly regulate the expression of multiple germ cell-specific genes, including Asz1 In addition, conditional ablation of NRF1 in gonocytes dramatically down-regulated these germline genes, blocked germ cell proliferation, and subsequently led to male infertility in mice. Our data highlight a precise crosstalk between transcriptional regulation by NRF1 and epigenetic modulation during germ cell development and unequivocally demonstrate a novel role of NRF1 in spermatogenesis.-Wang, J., Tang, C., Wang, Q., Su, J., Ni, T., Yang, W., Wang, Y., Chen, W., Liu, X., Wang, S., Zhang, J., Song, H., Zhu, J., Wang, Y. NRF1 coordinates with DNA methylation to regulate spermatogenesis.

Keywords: ASZ1; germline gene regulation; mitochondria.

Figure 1.

The expression of ASZ1 is regulated by DNA methylation. A) Asz1 promoter contains multiple CpG-rich sites. B) Transcript levels of ASZ1 in MEF and NIH3T3 cells in the absence or presence of 5′-Aza by RT-PCR. C) RT-PCR of Asz1 and β-actin on total RNAs or MSRE-PCR on the promoter region of Asz1 (pAsz1) from various tissues. The deduction of methylation status and expression level of Asz1 was summarized in Table 1. No expression of Asz1 was detected when its promoter was methylated, which was indicated by a band present with prior treatment with HpaII. D) Bisulfate sequencing of the Asz1 promoter on genomic DNA. White circles: unmethylated CpG; black circles: methylated CpGs.

Figure 2.

NRF1 coordinates with DNA methylation to modulate ASZ1 expression. A) Asz1 core promoter contains 2 NRF1 binding sites. B) ChIP assays followed by real-time PCR analyses with a NRF1 antibody from various tissues on the Asz1 promoter containing predicated NRF1 binding sites. C, D) Gel shift assays of a GST-NRF1 fusion protein with synthesized FITC- or ³²P-labeled probe containing NRF1 binding sites 1 (C) and 2 (D), respectively. The GST-ASZ1 fusion protein was used as a negative control. E) Activities of luciferase driven by wild-type, mutated (with 1 or both NRF1 binding site deleted: e.g., pΔb1), or methylated promoter of Asz1 were measured upon cotransfection of a NRF1 transgene or an empty vector control (ctrl). The pGL3 is an additional vector control without Asz1 promoter. Relative firefly luciferase activity is normalized to the Renilla and represented as mean ± 1 sem from 6 biologic replicates. *P < 0.05, **P < 0.01, ***P < 0.001. F) Vector containing the Asz1 promoter (pASZ1 in E) was treated with or without CpG methyltransferase M.SssI (MpAsz1) and methylation status was confirmed by digestion of the methylation-sensitive restriction enzyme HpaII.

Figure 3.

NRF1 targets multiple germ cell–specific genes in testes. A) Luciferase activities driven by Lin28a or Ddx25 promoters containing NRF1 binding sites were examined upon cotransfection of an NRF1 transgene or a vector control (ctrl). Relative firefly luciferase activity was normalized to Renilla activity and represented as means ± 1 sem from 6 biologic replicates. ***P < 0.001. B) NRF1 is mainly enriched at promoter regions of its targeted genes. C) Confirmation of NRF1 target sites by ChIP-PCR with an antibody against NRF1 from wild-type adult testes. D) GC1 cells were treated with or without 5′-Aza and transfected with a vector control or a NRF1-expressing plasmid. Transcript levels of NRF1 target genes were measured by real-time RT-PCR and represented as mean ± 1 sem from 6 biologic replicates. *P < 0.05, **P < 0.01, ***P < 0.001. Minus indicates without; plus denotes in the presence of 5′-Aza or the NRF1 transgene.

Figure 4.

Establishing Nrf1 conditional-knockout ESCs and mouse model. A) NRF1 protein was examined by IHC. B) Transcript level of NRF1 was measured by real-time RT-PCR in CD9⁺/C-KIT⁻ spermatogonia, C-KIT⁺ spermatocytes, and haploid spermatids from wild-type adult testes. Data are means ± 1 sem from 4 biologic replicates. **P < 0.01. C) The NRF1 protein level was measured by Western blot and IHF in testes with conditional deletion of Nrf1 at P7, compared with their wild-type littermates. Arrowheads: the DDX4⁺ germ cells that were devoid of NRF1 expression in Nrf1^f/Δ-Cre testicle sections. Scale bars, 50 μm.

Figure 5.

Conditional deletion of Nrf1 leads to male infertility. A) Gross morphology of testes from mice at 7 wk after birth. B) Histologic images of testicle sections or epididymis from wild-type mice or mice with Nrf1 deletion were obtained at different time points after birth. C, D) TUNEL assays (C) or PCNA staining (D) were performed on testes from wild-type control and mice with conditional Nrf1 deletion at various postnatal time points. Scale bars, 50 μm, testicle sections; 100 μm, epididymis sections.

Figure 6.

NRF1 deficiency leads to reduced expression of germ cell–specific genes. A) Real-time RT-PCR analyses on the testes with conditional deletion of Nrf1 in germ cells at P5, compared to their wild-type littermates. Data are the mean of relative expression from 6 replicates ± 1 sem. **P < 0.01, *P < 0.05, ***P < 0.001. B) IHF of ASZ1 and TRA98 in testes from wild-type mice or mice with Nrf1 conditional deletion. Scale bar, 50 μm. C) IHF of germ cell–related proteins (DDX4, Lin28a, and DAZL) and HSP60 (a mitochondrial protein) in testes from wild-type mice or mice with Nrf1 conditional deletion. Scale bar, 10 μm. D) Protein levels of NRF1 target genes and genes involved mitochondrial activities measured by Western blots on the testes. E) Bisulfate sequencing of the Asz1 promoter from wild-type control and mice with Nrf1 deletion at P8. White circles, unmethylated CpGs; black circles, methylated CpGs.