Dicer, a new regulator of pluripotency exit and LINE-1 elements in mouse embryonic stem cells

Abstract

A gene regulation network orchestrates processes ensuring the maintenance of cellular identity and genome integrity. Small RNAs generated by the RNAse III DICER have emerged as central players in this network. Moreover, deletion of Dicer in mice leads to early embryonic lethality. To better understand the underlying mechanisms leading to this phenotype, we generated Dicer-deficient mouse embryonic stem cells (mESCs). Their detailed characterization revealed an impaired differentiation potential, and incapacity to exit from the pluripotency state. We also observed a strong accumulation of LINE-1 (L1s) transcripts, which was translated at protein level and led to an increased L1s retrotransposition. Our findings reveal Dicer as a new essential player that sustains mESCs self-renewal and genome integrity by controlling L1s regulation.

Keywords: Dicer; LINE‐1 retrotransposition; mouse embryonic stem cells; transposable elements.

Figure 1

Generation of Dicer_KO mESCs using the paired CRISPR/Cas9 approach. (A) CRISPR/Cas9 design. The structure of the DICER protein is shown at the top, with the genomic regions corresponding to area from the PAZ domain to the second RNAse III domain below. The anti‐DICER antibody recognizes the 961–975 amino acids region of the PAZ domain. Three CRISPR/Cas9 single guide RNAs (sgRNAs), targeting the Dicer gene were designed: sgRNA 1 in the exon 16, sgRNA 2 in‐between exon 22 and 23, and sgRNA 3 in‐between exon 23 and 24. The combination of the sgRNAs 1 and 3 deleted the region between the PAZ domain and the second RNAse III domain (Δ13), and the sgRNAs 2 and 3 erased the second catalytic RNase III domain (Δ23). Specific genotyping primers have been designed around each sgRNA‐binding sites allowing a PCR screening of positive candidates for the deletions, used in (B). (B) PCR on genomic DNA of WT and Dicer_KO mESCs. Deletions Δ23 and Δ13 were confirmed by the presence of DNA amplicons of 413 bp and 492 bp, respectively. (C) Immunoblot analysis of DICER protein levels in WT and Dicer_KO mESCs. For protein normalization, α‐Tubulin (TUB) was used as a loading control. Representative blot of three independent experiments is shown. (D) Northern blot analysis using WT and Dicer_KO mESCs total RNA extract probed with specific miR‐295 and miR‐16 probes. Pre‐miRNA and mature miRNAs are indicated by arrows. Samples were probed with a U6‐specific probe as loading control. Representative blot of three independent experiments is shown. (E) Immunoblot analysis of DICER, DROSHA, DGCR8, AGO2, and AGO1 protein levels in WT and Dicer_KO mESCs. For protein normalization, α‐Tubulin (TUB) was used as a loading control. Representative blot of three independent experiments is shown. (F) Volcano plot showing the global transcriptional changes in Dicer_KO vs WT mESCs. Each circle represents one gene. The x‐axis shows the log fold change and the y‐axis shows the log10 of the P‐value. Differentially expressed genes are represented by colored circles and are defined by a fold change superior to 2 and a false discovery rate inferior to 0.01. (G) Graphical demonstration of associated biological processes of differentially expressed genes in Dicer_KO relative to WT mESC samples. The y‐axis displays the biological process categories that are identified in the analysis. The x‐axis shows the enrichment score, which is the value of −log10(P‐value). Functions are listed from the most enriched to least. The top 20 biological process categories are displayed. Pathways analysis has been performed using the Consensus PathDB‐mouse database (CPDB) 93, 94.

Figure 2

Characterization of newly generated Dicer_KO mESCs. (A) Proliferation assay of WT and Dicer_KO mESCs. For each cell line, data are shown as the fold change in the number of metabolically active cells compared to the first measurement done 24 h after the plating. Data are represented as mean ± SD (n = 3). (B) Cell cycle analysis of WT and Dicer_KO mESCs. Data are represented as mean ± SD (n = 3). (C) Apoptotic cell population analysis of WT and Dicer_KO mESCs. Data are represented as mean ± SD (n = 3). (D) Visualization of WT and Dicer_KO mESCs at Day 0 (upper panel) and at Day 10 (lower panel) of embryoid body (EB) differentiation. Scale bar = 50 μm. (E) RT‐qPCR analysis of three ectoderm markers: Pax6, Nestin, and Fgf5 mRNAs in WT and Dicer_KO mESCs. The data are shown as the fold change compared to WT cells after normalization to the Gapdh housekeeping gene at Day 0. Data are represented as mean ± SD (n = 3). (F) RT‐qPCR analysis of three endoderm markers: Dab2, Gata6 and Gata4 mRNAs in WT and Dicer_KO mESCs. The data are shown as the fold change compared to WT cells after normalization to the Gapdh housekeeping gene at Day 0. Data are represented as mean ± SD (n = 3). (G) RT‐qPCR analysis of three ectoderm markers: Fgf8, Brachyury, and Actc1 mRNAs in WT and Dicer_KO mESCs. The data are shown as the fold change compared to WT cells after normalization to the Gapdh housekeeping gene at Day 0. Data are represented as mean ± SD (n = 3). (H) RT‐qPCR analysis of pluripotency markers: Oct4 (Pou5f1), Nanog, and Sox2 mRNAs in WT and Dicer_KO mESCs before and after 10 days of EB differentiation. The data are shown as the fold change compared to WT cells after normalization to the Gapdh housekeeping gene at Day 0. Data are represented as mean ± SD (n = 3). (I) Immunoblot analysis of OCT4, NANOG, and SOX2 protein levels in WT and Dicer_KO mESCs at Day 0 and Day 10 of EB differentiation. For protein normalization, α‐Tubulin (TUB) was used as a loading control. L = Protein Ladder. Representative blot of three independent experiments is shown.

Figure 3

Dicer is essential for mESCs to exit from the pluripotent state. (A) Schematic design of the exit from pluripotency experiment. (B) Left panel corresponds to the visualization of WT and Dicer_KO mESCs after the alkaline phosphatase (AP) staining: full six‐well plate (scale bar = 1 cm) and magnified (scale bar = 50 μm). Representative pictures of three independent experiments are shown. Right panel displays the clonal AP quantification from whole well pictures from three independent exit from pluripotency assays. The data are shown as the number of AP positives colonies counted. Data are represented as mean ± SD (n = 3). (C) Flow cytometry analysis of pluripotent factors OCT4 and NANOG coexpression in WT and Dicer_KO mESCs in serum + LIF condition. Representative analysis of three independent experiments. (D) Flow cytometry analysis of transcription factors OCT4 and NANOG in WT and Dicer_KO mESCs in 2i condition. Representative analysis of three independent experiments. (E) Flow cytometry analysis of pluripotent factors STELLA and SSEA‐1 coexpression in WT and Dicer_KO mESCs in serum + LIF condition. Representative analysis of three independent experiments.

Figure 4

LINE‐1 elements are strongly up‐regulated in Dicer_KO mESCs. (A) Boxplot representing the log2 of Reads Per Kilobase per Million (RPKM) of the three major retrotransposon subclasses in WT and Dicer_KO mESCs. As a control, we used satellite repeats, which do not belong to the transposable element (TE) family. Statistical analysis has been performed using a two‐tailed t‐test. n.s., not significant, *P‐value < 0.05, ***P‐value < 0.005. (B) RT‐qPCR analysis of two LTR types IAP, MuERV‐L and SINE mRNAs in WT and Dicer_KO mESCs. The data are shown as the fold change compared to WT cells after normalization to the Gapdh housekeeping gene. Data are represented as mean ± SD (n = 3). (C) Schematic representation of a murine L1. A full active element is about 7 kb in length and composed of a 5′UTR, two ORFs, and a 3′UTR. In mice, three active L1s subfamilies can be distinguished: Tf, Gf and A 26, 27, 95, which are defined by the variable sequence and numbers of monomers (tandem repeat units of 200 bp) contained in their 5′UTR 96. RT‐qPCR primers for overall L1s expression assessment have been designed in ORF2, and specific RT‐qPCR primers for each L1s subfamily have been designed in the 5′UTR, used in (D). (D) RT‐qPCR analysis of overall L1s and specific L1 subfamily mRNAs in WT and Dicer_KO mESCs. The data are shown as the fold change compared to WT cells after normalization to the Gapdh housekeeping gene. Data are represented as mean ± SD (n = 3). (E) Immunoblot analysis of L1_ORF1 protein levels in WT and Dicer_KO mESCs. For protein normalization, α‐Tubulin (TUB) was used as a loading control. Representative blot of three independent experiments are shown.

Figure 5

DICER restricts LINE‐1 retrotransposition in mESCs. (A) Northern blot analysis using WT and Dicer_KO mESCs total RNA extract probed with a specific L1_probe. Full‐length L1s transcripts are indicated with an arrow. Ethidium bromide staining before transfer was used to confirm equal loading. 28S RNA is shown as a loading control. (B) Description of the different plasmids used for the L1 EGFP‐based retrotransposition assay. (C) Schematic representation of the L1 EGFP‐transgene and its retrotransposition (adapted from 73). The L1‐EGFP transgene (pL1_RP) consists of a human L1_RP element driven by the mouse RNA pol II promoter in addition to its endogenous 5′UTR. This element is coupled to an EGFP gene directed in the antisense orientation and interrupted by the mouse γ‐globin intron in the same transcriptional orientation as the L1. Therefore, when the L1‐EGFP transgene transcript is processed, the mouse γ‐globin intron is spliced out and the EGFP gene can be expressed after reverse transcription and integration into the genomic DNA. In the case of retrotransposition events, mESCs will express EGFP. In the negative control (pL1_{JM 111}), the L1_RP element has been replaced by the L1_{JM 111} element. The L1_{JM 111} element is a nonfunctional L1 transgene consisting in a human L1 mutated in ORF1 (*) 70, abrogating its retrotransposition activity. (D) Retrotransposition assay experiment design and time line in mESCs. (E) Flow cytometry gating strategy for the analysis of GFP‐positive cells in (F). We first selected the mESC population and subsequently excluded the doublets in both dimensions. The data from the first triplicate of Δ13 mESCs transfected with the pL1_RP (week 6) plasmid were used to represent the gating strategy. The gating for EGFP‐positive and EGFP‐negative cells was determined by analyzing cells transfected with: a plasmid coding EGFP and a puromycin‐resistance gene and a plasmid coding only a puromycin‐resistance gene respectively described in (B). 3.10⁴ events per samples were set as a final gate. (F) Histograms summarizing the FACS analysis of the retrotransposition of pL1_RP and the pL1_{JM 111} transgenes in WT and Dicer_KO mESCs at week 3 and week 6 after transfection. The data are shown as percentage of GFP‐positive cells. Data are represented as mean ± SD (n = 3).

Figure 6

Original plots from the L1 retrotransposition assay FACS analysis. GFP‐positive cells gating strategy used for the FACS analysis of the plots generated during the L1 retrotransposition assay (FSC‐W vs GFP‐A). Cells were first gated for living population (SSC‐A vs FSC‐A) and then gated for single events (FSC‐H vs FSC‐W) and (SSC‐H vs SSC‐W). Plots for each experiment are shown in A, B, and C. L1 retrotransposition assay was performed in triplicate. (A) First triplicate. (B) Second triplicate. (C) Third triplicate.