Protein Arginine Methyltransferase 6 (Prmt6) Is Essential for Early Zebrafish Development through the Direct Suppression of gadd45αa Stress Sensor Gene

Abstract

Histone lysine methylation is important in early zebrafish development; however, the role of histone arginine methylation in this process remains unclear. H3R2me2a, generated by protein arginine methyltransferase 6 (Prmt6), is a repressive mark. To explore the role of Prmt6 and H3R2me2a during zebrafish embryogenesis, we identified the maternal characteristic of prmt6 and designed two prmt6-specific morpholino-oligos (MOs) to study its importance in early development, application of which led to early epiboly defects and significantly reduced the level of H3R2me2a marks. prmt6 mRNA could rescue the epiboly defects and the H3R2me2a reduction in the prmt6 morphants. Functionally, microarray data demonstrated that growth arrest and DNA damage-inducible, α, a (gadd45αa) was a significantly up-regulated gene in MO-treated embryos, the activity of which was linked to the activation of the p38/JNK pathway and apoptosis. Importantly, gadd45αa MO and p38/JNK inhibitors could partially rescue the defect of prmt6 morphants, the downstream targets of Prmt6, and the apoptosis ratios of the prmt6 morphants. Moreover, the results of ChIP quantitative real time PCR and luciferase reporter assay indicated that gadd45αa is a repressive target of Prmt6. Taken together, these results suggest that maternal Prmt6 is essential to early zebrafish development by directly repressing gadd45αa.

Keywords: apoptosis; c-Jun N-terminal kinase (JNK); gadd45αa; histone methylation; p38; prmt6; zebrafish.

FIGURE 1.

Prmt6 is expressed throughout early zebrafish development. A, a representative Western blot showing the level of H3R2me2a in normal embryos during early zebrafish development with H3 and β-actin as loading controls. -Fold of basal, the H3R2me2a ratios were determined by the densitometric value of each treatment relative to that of the control (1.25 hpf) after normalizing to the H3 densitometric values. Protein load per lane, 40 μg. B, a representative agarose gel showing the mRNA expression level of prmt6 in unfertilized eggs (UE) and during early zebrafish development with β-actin as a loading control. C, the expression pattern of prmt6 was also determined by whole mount in situ hybridization at 0.2 (lateral view), 2 (lateral view), 3 (lateral view), 4.5 (lateral view), 6 (lateral view), and 10 hpf (lateral view with dorsal toward right). Scale bar, 200 μm.

FIGURE 2.

Prmt6 is essential for early zebrafish development. A, successful suppression of prmt6 expression was confirmed in embryos injected with the plasmid pCS2+-prmt6-5′-UTR-GFP and prmt6 MO1/MO2. B, knockdown of Pmrt6 expression using the prmt6 MOs resulted in a normal, mild, or severe phenotype characterized by defective epiboly as shown at 6 hpf. The single enlarged embryo is shown from lateral views with the animal pole on the top. C, a quantification of the relative distribution of the phenotype of prmt6 morphants at 6 hpf. D, a quantification of the relative distribution of the phenotype of prmt6 morphants rescued with myc-prmt6 mRNA (containing only the coding sequence) and mutated myc-prmt6 mRNA (containing only the coding sequence) that codes an inactive form of Prmt6 (VLD to KLA). E, a representative Western blot showing the protein expression level of the rescue constructs by Myc tag. -Fold of basal, the Myc ratios were determined by the densitometric value of each construct relative to that of the control (myc-prmt6 mRNA) after normalizing to the β-actin densitometric values. myc-prmt6 mRNA/myc-prmt6 mutated mRNA, 200/200 pg. F, a representative Western blot showing the effect of the prmt6 MOs on the H3R2me2a level. cMOs/MOs, 0.3/0.3 mm; myc-prmt6 mRNA/myc-prmt6 mutated mRNA, 200/200 pg. G, a quantification of the relative distribution of the phenotype of prmt6 morphants and a representative Western blot showing the p53 protein level rescued with p53 MO. Protein loads per lane for E, F, and G, 37.5 μg. In C, D, and G, the number of embryos in each group is indicated above the relevant column. Scale bar in A and B, 200 μm.

FIGURE 3.

Inhibition of the up-regulation of gadd45αa partially rescues the prmt6 morphants. A, a microarray analysis of the prmt6 morphants at 6 hpf was conducted, and the top 10 up-regulated and down-regulated genes are shown. B, microarray data were validated by RT-qPCR, confirming that prmt6 MO1 induced the expression of gadd45αa, hsp70, tbpl2, and rx3 and decreased the expression of eve1 and sox2. C, a gadd45αa MO was designed and validated in embryos injected with the plasmid pCS2+-gadd45αa-5′-UTR-GFP and the gadd45αa MO. D, a splice-blocking tbpl2 MO was designed and validated according to the strategy described under “Experimental Procedures” with β-actin as a loading control. Co-injection of a gadd45αa MO partially rescues the prmt6 morphants in a dose-dependent manner (E), whereas co-injection of hsp70 MO or tbpl2 MO has no obvious effects on the prmt6 morphants (F). G, interestingly, a quantification of the relative distribution of embryonic phenotypes at 6 hpf showed that overexpression of gadd45αa phenocopies the prmt6 morphants. In E, F, and G, the number of embryos in each group is indicated above the relevant column. Scale bar in C, 200 μm. Error bars represent S.D.

FIGURE 4.

The p38/JNK pathway is activated in the prmt6 morphants. A, downstream targets of the p38/JNK pathway (c-jun, p53, and p21) were up-regulated in the prmt6 morphants. B, the extent of apoptosis was measured in the prmt6 morphants by flow cytometry. C, co-injection with inhibitors of the p38/JNK pathway partially rescued the prmt6 morphants. The inhibition of the phosphorylation of c-Jun by SP600125 was shown by Western blotting. Representative quantitative analyses of targets of Prmt6 by RT-qPCR following rescue are shown (D and E). F, in agreement with the above data, the prmt6 mRNA, gadd45αa MO, and p38/JNK pathway inhibitors decreased the ratios of early apoptosis (Annexin V+/PI−) in the prmt6 morphant, whereas no obvious effects of the prmt6 mutated mRNA and p53 MO were observed. In C, the number of embryos in each group is indicated above the relevant column. Protein load per lane, 37.5 μg. Error bars represent S.D.

FIGURE 5.

Gadd45αa is a direct target of Prmt6. A, a representative Western blot showing the protein expression level of the myc-prmt6 mRNA using Myc tag. myc-prmt6 mRNA, 25 pg. Our ChIP data revealed that Prmt6 binds to the promoter of gadd45αa (B) but not tbpl2 (C). Furthermore, prmt6 MO1 decreases the modification of H3R2me2a on the promoter of gadd45αa (D) but increases the modification of H3K4me3 (E). F, in agreement with the above data, the luciferase activity assay demonstrated that prmt6 MO1 enhances the activity of the promoter of gadd45αa, whereas prmt6 mRNA represses the activity of the promoter of gadd45αa. Under H₂O₂ treatment, the transcription of gadd45αa was induced, the JNK pathway was activated (G), and the H3R2me2a level at the gadd45αa promoter (−107) was decreased (H) at 4 and 6 hpf. I, a diagram illustrating the requirement of H3R2me2a for early embryogenesis through preventing the activation of the p38/JNK pathway by directly suppressing gadd45αa. Protein load per lane for A and G, 37.5 μg. *, p < 0.05; **, p < 0.01. Error bars represent S.D. TSS, transcriptional start site.