Dual repression of endocytic players by ESCC microRNAs and the Polycomb complex regulates mouse embryonic stem cell pluripotency

Abstract

Cell fate determination in the early mammalian embryo is regulated by multiple mechanisms. Recently, genes involved in vesicular trafficking have been shown to play an important role in cell fate choice, although the regulation of their expression remains poorly understood. Here we demonstrate for the first time that multiple endocytosis associated genes (EAGs) are repressed through a novel, dual mechanism in mouse embryonic stem cells (mESCs). This involves the action of the Polycomb Repressive Complex, PRC2, as well as post-transcriptional regulation by the ESC-specific cell cycle-regulating (ESCC) family of microRNAs. This repression is relieved upon differentiation. Forced expression of EAGs in mESCs results in a decrease in pluripotency, highlighting the importance of dual repression in cell fate regulation. We propose that endocytosis is critical for cell fate choice, and dual repression may function to tightly regulate levels of endocytic genes.

Figure 1

In silico identification of transcription factors and transcriptional regulators that regulate endocytic gene expression in pluripotent and differentiated cells. (a) Bar chart displaying the statistical association between gene sets bound by different transcriptional regulators (from ChIP-seq binding data in Chen et al.), and the set of genes whose expression levels are 2-fold higher in MEFs relative to mESCs. Differentially upregulated genes are significantly over-represented in the gene targets of SUZ12 (indicated by a low p-value and high Z-score). (b) Bar chart showing the 50 genes with known association with endocytosis, whose promoter regions are bound by SUZ12 and are upregulated in MEFs relative to mESCs. The interaction score between SUZ12 and each gene, as well as the log2 fold expression change of each gene, are shown. (c) RT-qPCR analysis of SUZ12 bound endocytic genes in mESCs and MEFs. mRNA expression is normalized to Gapdh, and further normalized to expression in mESCs. Error bars represent mean ± S.D for experiments in triplicates (N = 3). *p < 0.05; **p < 0.01; ***p < 0.001 by Students T-test.

Figure 2

Dual regulation of endocytic genes by the PRC2 complex and ESCC miRNAs. (a) RT-qPCR analysis of PRC2 target genes upon Ezh2 knockdown in V6.5 and R1 mESCs. mRNA expression is normalized to Gapdh and represented relative to scrambled shRNA. Scrambled shRNA is represented as a dashed line at 1. (b) Graph showing enrichment of SUZ12 at Cav1, Cdh2, Tgfbr1, Tgfbr2 and Tgfbr3 promoters by chromatin immunoprecipitation using IgG or SUZ12 specific antibody. (c) RT-qPCR analysis for endocytic gene expression in MEFs in the presence of exogenous miR-294, 12 and 24 hrs post-transfection. Mock is represented as a dashed line at 1. (d) RT-qPCR analysis of PRC2 target genes in Dgcr8 KO mESCs upon Ezh2 knockdown, and in the presence of exogenous miR-294. mRNA expression is normalized to Gapdh and represented relative to scrambled shRNA. Scrambled shRNA is represented as a dashed line at 1. (e) Luciferase analysis of Cav1 3′UTR, Cdh2 (N-Cad) ORF and human Tgfbr2 3′UTR. MEFs were co-transfected in the presence or absence of miR-294 and respective pSICHECK2 vectors. Renilla luciferase activity levels were normalized to firefly luciferase, which served as an internal control. Mock transfection level is represented as a dashed line at 1. For all experiments, error bars represent mean ± S.D for experiments in triplicates (N = 3). *p < 0.05; **p < 0.01; ***p < 0.001 by Students T-test.

Figure 3

Misexpression of Cav1/Cdh2 in mESCs provide a differentiation cue in mESCs. (a) RT-qPCR analysis for Cav1 and Cdh2 mRNA expression in the following samples: mESCs cultured in the presence of LIF, mESCs cultured in the absence of LIF for 72hrs, mESCs cultured with retinoic acid for 96hrs, and in MEFs (N = 3). (b) Western blot showing expression of CAV1 and CDH2 in mESCs and MEFs (N = 3). (c–f) RT-qPCR analysis of pluripotency markers (c,e) and differentiation markers (d,f) in mESCs upon overexpression of Cav1, along with Cavin1 or Cdh2. For all experiments, error bars represent mean ± S.D for experiments in triplicates (N = 3). *p < 0.05; **p < 0.01; ***p < 0.001 by Students T-test. (g) Time-lapse confocal images of the equatorial plane showing ingression of the first cytokinetic furrow in control and cav-1(RNAi) C. elegans embryos co-expressing GFP::PLC1δ –PH (membrane marker) and Histone::mCherry (DNA marker). The furrow takes a longer time to complete ingression upon CAV-1 depletion. Inset shows the region of interest (ROI) made into a montage.

Figure 4

Dual repression of endocytic players in pluripotent mESCs. Model showing dual repression of endocytic players by SUZ12 (transcriptional repression at the promoter), and ESCC microRNAs (post-transcriptional regulation) in mESCs, resulting in a decreased expression of these genes. In differentiated cells, both these modes of repression are relieved, resulting in expression of these genes.