PRMT5 Is Involved in Spermatogonial Stem Cells Maintenance by Regulating Plzf Expression via Modulation of Lysine Histone Modifications - PubMed
- ️Fri Jan 01 2021
PRMT5 Is Involved in Spermatogonial Stem Cells Maintenance by Regulating Plzf Expression via Modulation of Lysine Histone Modifications
Fangfang Dong et al. Front Cell Dev Biol. 2021.
Abstract
Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of mono- or symmetric dimethylarginine residues on histones and non-histone substrates and has been demonstrated to play important roles in many biological processes. In the present study, we observed that PRMT5 is abundantly expressed in spermatogonial stem cells (SSCs) and that Prmt5 deletion results in a progressive loss of SSCs and male infertility. The proliferation of Prmt5-deficient SSCs cultured in vitro exhibited abnormal proliferation, cell cycle arrest in G0/G1 phase and a significant increase in apoptosis. Furthermore, PLZF expression was dramatically reduced in Prmt5-deficient SSCs, and the levels of H3K9me2 and H3K27me2 were increased in the proximal promoter region of the Plzf gene in Prmt5-deficient SSCs. Further study revealed that the expression of lysine demethylases (JMJD1A, JMJD1B, JMJD1C, and KDM6B) was significantly reduced in Prmt5-deficient SSCs and that the level of permissive arginine methylation H3R2me2s was significantly decreased at the upstream promoter region of these genes in Prmt5-deficient SSCs. Our results demonstrate that PRMT5 regulates spermatogonial stem cell development by modulating histone H3 lysine modifications.
Keywords: PLZF; PRMT5; histone lysine modification; lysine demethylase; spermatogonial stem cells.
Copyright © 2021 Dong, Chen, Chen, Jiang, Shen, Ma, Han, Guo and Gao.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures

Deletion of Prmt5 causes germ cell loss and male infertility. No obvious developmental abnormalities were observed in adult Prmt5Δ/flox;Mvh-Cre mice (A), and the body weights were comparable to those of control littermates (B). The size of testes from Prmt5Δ/flox;Mvh-Cre mice was significantly smaller than that of control littermates (C,D). Germ cells at different developmental stages were observed in control testes (E), whereas most seminiferous tubules were empty in adult Prmt5Δ/flox;Mvh-Cre testes (F, asterisks). The cauda epididymis of control males was filled with mature sperm (G), but no mature sperm were observed in the epididymis of Prmt5-deficient males (H). PLZF-positive spermatogonia were located at the peripheral region of the seminiferous tubules in control mice [(I), black arrows], whereas no PLZF-positive spermatogonia were observed in Prmt5Δ/flox;Mvh-Cre mice (J). SOX9-positive Sertoli cells were observed in the seminiferous tubules of both control [(K), black arrows] and Prmt5Δ/flox;Mvh-Cre mice [(L), black arrows]. Error bars represent the SEM of triplicate results. ****P < 0.00001 indicates a significant difference (t-test).

Germ cells from Prmt5Δ/flox;Mvh-Cre mice are gradually lost from P10. The germ cells in control and Prmt5Δ/flox;Mvh-Cre mice were labeled with an anti-MVH antibody. MVH-positive germ cells were observed in both control [(A), black arrows] and Prmt5Δ/flox;Mvh-Cre mice [(B), black arrows] at P7, and no difference was noted. The number of germ cells in Prmt5Δ/flox;Mvh-Cre mice [(D), black arrows] was significantly reduced at P10 compared to that observed in control mice [(C), black arrows]. The number of germ cells was significantly increased from 2 to 6 weeks in control mice [(E,G,I,K), black arrows]. In contrast, the number of germ cells in Prmt5Δ/flox;Mvh-Cre mice was gradually reduced from 2 to 4 weeks [(F,H,J), black arrows] and was completely absent at 6 weeks of age [(L), asterisks].

Prmt5-deficient germ cells exhibit a defect in meiosis initiation. A strong STRA8 signal was detected in the germ cells of control testes [(A), white arrows], whereas only a weak STRA8 signal was detected in the germ cells of Prmt5Δ/flox;Mvh-Cre testes [(B), white arrows]. SYCP3-positive germ cells were observed in control testes [(C), white arrows], but no SYCP3 signal was detected in the germ cells of Prmt5Δ/flox;Mvh-Cre testes [(D), asterisks]. The expression of meiosis-associated genes (STRA8, SYCP3 and SYCP1) was significantly reduced in Prmt5Δ/flox;Mvh-Cre testes at P10 (E,F), The total protein detected from control testes was 10 μg, and the total protein detected from Prmt5Δ/flox;Mvh-Cre testes was 40 μg. MVH was used as a loading control in (E), protein values were normalized to MVH and expressed as the mean ± SEM (n = 3), *P < 0.01, **P < 0.001 indicates a significant difference (t-test).

Prmt5-deficient spermatogonial stem cells exhibit abnormal proliferation. Ki67-positive (red) germ cells were detected in both control [(A), white arrows] and Prmt5Δ/flox;Mvh-Cre testes [(B), white arrows] at P10. The percentage of Ki67-positive germ cells was dramatically reduced in Prmt5Δ/flox;Mvh-Cre mice (C). PH3 (red) was detected in MVH-positive germ cells of both control [(D), white arrows] and Prmt5Δ/flox;Mvh-Cre testes [(E), white arrows] at P10. The percentage of PH3-positive germ cells was dramatically reduced in Prmt5Δ/flox;Mvh-Cre mice (F). (G) Bright-field images of control and Prmt5-deficient SSCs cultured in vitro. Prmt5flox/flox;Cre-ERTM SSCs were treated with ethanol or 1 μM tamoxifen to induce Cre activation. (H) MTT assay of cultured control and Prmt5-deficient SSCs. (I) FACS analysis of cultured control and Prmt5-deficient SSCs. (J) Quantitative analysis of cells in G0/G1, S, and G2 phase. Error bars represent the SEM of triplicate results. *P < 0.05, **P < 0.005, ***P < 0.0005 indicates a significant difference (t-test).

Differentially expressed genes in Prmt5-deficient spermatogonial stem cells. The expression of genes related to self-renewal and proliferation was analyzed by real-time PCR (A) and western blotting (B) in Prmt5flox/flox;Cre-ERTM SSCs treated with ethanol or 1 μM tamoxifen. The quantitative results of western blot analysis (C). The expression of cell cycle-associated genes was analyzed by real-time PCR (D) and western blot (E). The quantitative results of western blot analysis (F). GAPDH was used as a loading control, and the protein values were normalized. Error bars represent the SEM of triplicate results. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0005 indicates a significant difference (t-test).

H3K9me2 and H3K27me2 levels are significantly increased in Prmt5-deficient spermatogonial stem cells. (A) The level of histone modification in control and Prmt5-deficient spermatogonial stem cells was examined by western blot. (B) Quantitative results of western blot analysis. ChIP assays were performed to analyze the enrichment of H3K27me2 (C) and H3K9me2 (D) modifications in the promoter regions (TSS-1 kb upstream of TSS) of the Plzf (site 1∼ site 5) gene in control and Prmt5-deficient SSCs. The relative enrichment of the Plzf promoter region was examined by real-time PCR using sequence-specific primer sets. IgG was used as a negative control. Site 1: −114∼−284 bp, Site 2: −175∼−347 bp, Site 3: −381∼−552 bp, Site 4: −573∼−743 bp, Site 5: −755∼−948 bp. Quantitative data are presented as the enrichment of the ChIP to the input DNA. TSS, transcription start site. Error bars present SEM of three ChIP experiments. *P < 0.05, **P < 0.005 indicates a significant difference (t-test).

The expression of lysine demethylases of H3K9me2 and H3K27me2 is regulated by PRMT5 via histone arginine modifications. The differential expression of lysine methylases and demethylases of H3K9me2 and H3K27me2 in Prmt5-deficient SSCs was analyzed by real-time PCR (A,B) and western blot (C,D). β-Actin was used as a loading control, and the protein values were normalized. ChIP-qPCR assays were performed to examine the enrichment of H4R3me2s or H3R2me2s modifications at the upstream regions of JMJD1A (E,I), JMJD1B (F,J), JMJD1C (G,K), and KDM6B (H,L) in Prmt5-deficient SSCs. The immunoprecipitated DNA fragments were analyzed by real-time PCR with sequence-specific primer sets. IgG was used as a negative control. Quantitative data are presented as the enrichment of the ChIP to the input DNA. Error bars represent the SEM of triplicate results. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005 indicates a significant difference (t-test).

Model of PRMT5 regulating Plzf expression via histone modifications in SSCs. In SSCs, PRMT5 promotes the expression of demethylases for H3K9me2 and H3K27me2 by catalyzing permissive histone H3R2me2s. The high expression of the corresponding lysine demethylases leads to low levels of H3K9me2 and H3K27me2 at the upstream promoter of Plzf, which in turn leads to the upregulation of the Plzf gene.
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