Pumilio proteins utilize distinct regulatory mechanisms to achieve complementary functions required for pluripotency and embryogenesis

Abstract

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.

Keywords: Pumilio; embryogenesis; mouse; stem cell; translational regulation.

Fig. 1.

Pumilio proteins are required for embryogenesis and normal growth. (A) Immunofluorescence staining of Pum1 in wild-type ESCs. Pum1 is diffusely cytoplasmic and is enriched in the nuclear periphery of some mitotic cells (arrows). Nuclei were counterstained with DAPI (blue). (B) Immunofluorescence staining of e2.5 morulae (Upper) and e3.5 blastocysts (Lower). Pum1 is expressed in both inner cell mass (ICM) and trophoblast (TB) cells, with greater cytoplasmic expression levels in the ICM. (C and D) Phenotype of P28 (C) and 11-mo-old (D) wild-type (wt), Pum1^+/−, and Pum1^−/− littermates. Pum1^−/− mice have a hunched appearance that becomes more prominent with age and weigh 43% less than wild-type mice (D, Upper Right). (E and F) Body weight (in grams) of wild-type (solid line), Pum1^+/− (dashed line), and Pum1^−/− (dotted line) littermates at P1, P7, P14, P21, and P28. P28 Pum1^−/− mice weigh 35% less than wild-type mice. Error bars indicate SEM. *P value < 0.01. (G) Deviation of the ratio of observed from expected genotypes of a Pum1^+/−; Pum2^+/− x Pum1^+/−; Pum2^+/− mating at e3.5 (dark gray, n = 52), e8.5 (light gray, n = 36), e9.5 (pink, n = 21), e12.5 (red, n = 23), and P1 (dark red, n = 127). The observed ratio of Pum1^−/−; Pum2^+/− pups is 65% less than expected at P1. No Pum1^−/−; Pum2^−/− pups were recovered at e9.5, e12.5, and 1dpp. * and ***indicates P < 0.01 and <0.001, respectively. (H) Morphology and average weight (in grams) of P1 wild-type, Pum1^+/−; Pum2^−/−, and Pum1^−/−; Pum2^+/− littermates. Pum1^+/−; Pum2^−/− pups weigh 29% less than WT littermates; Pum1^−/−; Pum2^+/− pups weigh 38% less than WT littermates, have no milk in their stomachs, and die within 24 h of birth. (I) Phenotype of P28 Pum-deficient mice; Pum1^−/− mice are smaller than Pum2^−/− mice and Pum1^+/−; Pum2^+/− mice, which are both indistinguishable from wild type at this age.

Fig. 2.

Pum1^−/−; Pum2^−/− mice are developmentally delayed and embryonic lethal by e8.5. Immunofluorescence staining of wild-type and mutant embryos for Oct4 (red) and endoderm marker Gata4 (green). (A and B) The e3.5 mutant embryos have no (or a smaller) blastocoel cavity and lack Gata4-positive primitive endoderm (PrE) cells. (C and D) The e4.5 mutant embryos have randomly positioned PrE cells in the ICM. (E and F) The e5.5 wild-type embryos develop an inner layer of Oct4-positive epiblast cells and an outer layer of visceral Gata4-positive endoderm cells. In mutant embryos, Gata4-positive PrE cells remain next to the blastocoel cavity. Nuclei were stained with DAPI (blue). (Scale bars, A–F, 25 µm.) (G) The e8.5 embryonic and extraembryonic tissues from Pum1^+/−; Pum2^+/− mated mice. Yolk sacs were used for genotyping. A Pum1^−/−; Pum2^−/− embryo (far right) is smaller than all littermates. (Scale bar, 1 mm.) (H–K) Higher magnification of dissected e8.5 embryos; the Pum1^−/−; Pum2^−/− double-knockout embryo (Inset in J, magnified in K) shows developmental delay, a primitive head fold, and overall lack of tissue with especially thin neural tissue. (Scale bars, H–K, 500 µm.)

Fig. 3.

Pum1; Pum2 double knockout (DKO) ESCs have decreased self-renewal capacity. (A and B) Alkaline phosphatase (AP) staining of ESCs cultured under self-renewal and differentiation conditions as viewed by low (A) and high (B) magnifications. (C–F) Quantification of AP staining in different culture conditions. Percentage of cell-colony numbers are shown as mean ± SD. t-test: *P < 0.05, **P < 0.01. (G and H) Western blot analysis of pluripotency markers in wild-type, Pum1^−/−, Pum2^−/−, and Pum1^−/−; Pum2^−/− ESCs cultured under self-renewal (G) and differentiation (H) conditions. Boxes highlight lower protein levels of Nanog (blue), Sox2 (red), and Oct4 (green) in Pum1^−/−; Pum2^−/− ESCs compared to wild type, lower levels of Nanog (blue) in Pum1^−/− cells compared to wild type, and higher levels of all three pluripotency markers in Pum1^−/− ESCs compared to wild type.

Fig. 4.

Pum1; Pum2 DKO ESCs have abnormal expression of key germ-layer differentiation markers in EBs and teratomas. (A) Morphological comparison of wild-type and mutant EBs on day 2, day 6, day 10, and day 12. (Scale bars, 250 μm.) (B–J) Relative expression of markers for pluripotency and three germ layers as quantified by qRT-PCR and normalized over GAPDH mRNA. Results are presented as mean ± SD. t-test: *P < 0.05, **P < 0.01, ***P < 0.001. (K) H&E staining of teratoma histological sections: (a) ectoderm-like pattern; (b) mesoderm-like pattern; (c) endoderm-like pattern.

Fig. 5.

Pum1 and Pum2 autoregulate each other and bind overlapping sets of functionally related mRNAs in ESCs. (A) Pum1 and Pum2 participate in an interregulatory loop (Upper); Pum1 and Pum2 mRNA have multiple putative PRE sites in their 3′ UTR (Middle: red lines, strict canonical TGTANATA motif; black lines, degenerate TDTANAWH motif). Western blotting analysis reveals increased Pum2 levels in Pum1^−/− ESCs (Lower Left) and increased Pum1 levels in Pum2^−/− ESCs (Lower Right). (B) Scatter plot of the log intensity of IP samples (y axis) and of negative controls (incubated with blocking peptide against which the antibody was raised [x axis]). (C) Heat map of enrichment in IP and control samples for the top 47 gene probes. (D) qRT-PCR of known Pum1 mRNA targets confirming enrichment in IP samples; error bars indicate SEM of three replicates. (E) GSEA of top 500 Pum1 mRNA targets by fold enrichment and P value. (F) Venn diagram indicating total number of significantly enriched (P < 0.001, fold enrichment >1.5) mRNA targets of Pum1 (pink) and Pum2 (green). (G) GSEA of the 354 overlapping mRNA targets of Pum1 and Pum2.

Fig. 6.

Pum1 and Pum2 regulate the translation of many mRNAs in ESCs. (A) The metaplot of ribosomal P-site mapping around the start and stop codons of annotated protein-coding regions in wild-type ESCs. The trinucleotide periodicity feature of ribosomal protection assay is clearly evident. +20-bp, −40-bp region relative to start codon is displayed for translation starting region; +40-bp, −20-bp region relative to end codon is displayed for translation stopping region. (B) The metaplot of P-site mapping around the start and the stop codon of annotated CDs in wild-type (black and red) and Pum double-mutant (blue, green, and orange) ESCs. Reads were normalized to sequencing depth. (C) Venn diagrams of translationally increased and decreased genes in Pum1^−/−, Pum2^−/−, and Pum double mutants compared to wild type. (D–E) The proportion of direct target and PRE-containing mRNA isoforms that are in translationally increased (D) or decreased (E) in Pum1^−/−, Pum2^−/−, and Pum double mutants compared to wild type.

Fig. 7.

A model of Pumilio function in ESCs and embryogenesis. During early embryogenesis, pluripotent stem cells (blue) differentiate into daughter cells (purple). Pum2 promotes ESC self-renewal while Pum1 promotes ESC differentiation; both negatively regulate each other’s expression. In the Pum1 knockout, Pum2 is overexpressed, and pluripotency markers Nanog, Oct4, and Sox2 are elevated. In the Pum2 knockout, Pum1 is overexpressed; Nanog levels are decreased while there is early expression of the differentiation markers Gata6, FoxA2, Brachyury, GSC, and FGF5. In the Pum1/2 double knockout there are decreased levels of both pluripotency and differentiation markers, delayed development by e3.5, and defective germ-layer development that leads to embryonic lethality by e8.5. Overall, this model highlights the role of Pum1 in differentiation, Pum2 in self-renewal, and both collectively for normal embryogenesis.