Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency

Abstract

Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in derepression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway.

Figure 1.

Dynamic expression of Prmt5 in early embryos and ES cell derivation. (A) Expression of Prmt5 (red) at various stages of early development. Oct4-positive cells as well as Blimp1-GFP-positive founder PGCs in E6.5 epiblasts are denoted in green. Nuclei were counterstained with DAPI (blue). (EPI) Epiblast; (pTE) polar TE; (mTE) mural TE. Bar, 20 μm. (B) Prmt5 (red) is up-regulated in the ICM outgrowth during ES cell derivation in vitro. Oct4 (green) marks pluripotent cells. (TGC) Trophoblast giant cells. (C) Prmt5 mRNA expression remains unchanged in Oct4-positive cells in the course of ES derivation, based on single-cell RNA-Seq analysis (Tang et al. 2010).

Figure 2.

Effects of Prmt5 loss of function and detection of cytoplasmic Prmt5 in ES cells. (A) A representative display of six blastocyst outgrowths (OG) from a Prmt5^+/− intercross. Outgrowths 4–6 showed impaired ICM proliferation, and were verified as Prmt5^−/− by qPCR. Levels of Prmt5 transcript were normalized against Gapdh, and results are shown on a log2 scale. Bar, 20 μm. (B) Immunostainings of Prmt5 (red) in pluripotent ES, TS, and differentiated ES cells. Oct4 and Cdx2 are in green. Nuclei were counterstained with DAPI (blue). (Right panel) Western blot of cytoplasmic and nuclear fractions from ES cells confirms cytoplasmic location of Prmt5. Purity of the preparations was confirmed by Oct4 (nuclear) and Tubulin (cytoplasmic) expression. Bar, 20 μm.

Figure 3.

Prmt5 is required for ES cell pluripotency. (A) Western blots depicting efficiencies of Prmt5 shRNA knockdown constructs (KD 1-4) compared with control (pSuper Control). Note phenotypic changes, diminution of Oct4 and Nanog, and alkaline phosphatase staining in Prmt5-depleted cells 5 d after knockdown. Tubulin was used as loading control. Bar, 30 μm. (B) qPCR analysis of gene expression in Prmt5 knockdown cells harvested 5 d after knockdown. Levels of transcripts were normalized relative to control shRNA transfection. Error bars indicate standard deviations of three technical replicates. (C) Microarray heat map depicting expression changes of selected ES-associated genes as well as developmental genes after Prmt5 knockdown (P < 0.01). Biological triplicates were used. (PKD) Prmt5 knockdown; (CTRL) control knockdown.

Figure 4.

Prmt5 methylates predeposited histone H2A. (A) In vitro methyltransferase assay using Flag-Prmt5 immunoprecipitated from ES cells and incubated with histone substrates (recombinant H2A, rH2A, or calf thymus histone preparations) and radioactive ³H-SAM. Fluorograph (FG) and Coomassie (CM) blots are shown. (B) Western blot showing loss of H2AR3me2s in Prmt5-depleted cells. (Left panel) Histones were isolated using acid extraction at day 5 of knockdown. ES cell fractions show H2AR3me2s/H4R3me2s in nuclear fraction but only H2AR3me2s in the cytoplasm (long Western blot exposure was performed). Purity of the preparations was confirmed by TBP (TATA-binding protein) (nuclear) and Tubulin (cytoplasmic) expression. (Right panel) H4R3me2a (asymmetric H4 dimethylation). (C) Time-course experiment showing loss of cytosolic H2AR3me2s upon depletion of Prmt5 in ES cells between 0 and 5 d. H4K5 acetylation (H4K5Ac) was used as a marker for cytosolic H4 and also served as loading control. Cytosolic H2AR3me2s was down-regulated first on day 3 (d3), concomitantly with the loss of Prmt5. Nuclear H2AR3me2s was subsequently down-regulated on day 5 (d5). (D) qPCR analysis of gene expression changes upon overexpression of methylation-deficient H2A and H4 histones. Mutant histones (containing IRES-GFP) were transfected into ES cells, and both “GFP⁺” and “GFP⁻” (control) cells were isolated by FACS and subjected to qPCR. Error bars represent the standard deviations of three technical replicates.

Figure 5.

Prmt5/Mep50-mediated H2AR3me2s is important for pluripotency. (A) Down-regulation of Mep50 during differentiation of ES cells. Loss of Mep50 at day 4 (d4) of differentiation was accompanied by a specific decrease in H2AR3me2s. (B) Prmt5 and Mep50 interact in ES cells. Immunoprecipitations using ES cells either transiently transfected with Flag-Prmt5 or stably expressing Prmt5-Flag (clone 9) and blotted for endogenous Mep50. Immunostainings showing colocalization of Mep50 with Flag-Prmt5 in the cytoplasm of ES cells. Transfected cells are denoted by GFP staining. Nuclei were counterstained with DAPI (blue). Bar, 20 μm. (C) Mep50 knockdown (5 d) leads to loss of H2AR3me2s and results in ES cell differentiation. Bar, 30 μm. (D) qPCR analysis of gene expression upon Mep50 knockdown (5 d) in ES cells. Levels of transcripts were normalized with respect to control shRNA transfection. Error bars indicate standard deviations of three technical replicates.