Opposing microRNA families regulate self-renewal in mouse embryonic stem cells

Abstract

When embryonic stem cells (ESCs) differentiate, they must both silence the ESC self-renewal program and activate new tissue-specific programs. In the absence of DGCR8 (Dgcr8(-/-)), a protein required for microRNA (miRNA) biogenesis, mouse ESCs are unable to silence self-renewal. Here we show that the introduction of let-7 miRNAs-a family of miRNAs highly expressed in somatic cells-can suppress self-renewal in Dgcr8(-/-) but not wild-type ESCs. Introduction of ESC cell cycle regulating (ESCC) miRNAs into the Dgcr8(-/-) ESCs blocks the capacity of let-7 to suppress self-renewal. Profiling and bioinformatic analyses show that let-7 inhibits whereas ESCC miRNAs indirectly activate numerous self-renewal genes. Furthermore, inhibition of the let-7 family promotes de-differentiation of somatic cells to induced pluripotent stem cells. Together, these findings show how the ESCC and let-7 miRNAs act through common pathways to alternatively stabilize the self-renewing versus differentiated cell fates.

Figure 1

The let-7 and ESCC miRNA families have opposing roles in regulating ESC self-renewal. (a) Transfected miRNAs with the seed sequence highlighted. (b) Pou5f1/Oct4 immunofluorescence staining after transfection of let-7c, miR-294 and combinations of let-7c with miR-294, mutant-miR-294, miR-291a-5p, or miR-130b in Dgcr8 -/- (i) and wild-type (ii) ESCs. Representative images, n = 3. (c) qRT-PCR for Pou5f1/Oct4, Sox2, and Nanog normalized to beta-actin after miRNA introduction as in b. n = 3-8. * indicates p < 0.02. (d) Colony reforming assays after miRNA introduction as in b and c. n = 3. * indicates p < 0.05. All p-values generated by Bonferroni corrected t-test of comparisons to let-7c treated. Error bars represent standard deviation.

Figure 2

The let-7 and ESCC miRNAs suppress hundreds of transcripts by binding their ORF and/or 3′UTR. (a) Microarray analysis following introduction of let-7c alone. Upregulated transcripts are shown in dark grey, downregulated transcripts in black (FDR < 0.05). (b) Analysis of seed matches in the promoter, 5′UTR, ORF, and 3′UTR of let-7c-downregulated and upregulated transcripts. Presented are the mean number of seeds matches per kb of sequence for the listed groups of altered genes described in a. P-values calculated by the Wilcoxon Rank Sum Test and Bonferroni corrected are shown for p < 0.01 . (c) Microarray analysis following introduction of miR-294 alone. Color labeling, as in a. (d) Seed analysis as in b for miR-294 up and downregulated transcripts.

Figure 3

Enrichment/depletion of transcription factor bound genes among miRNA-regulated transcripts. (a) A schematic of hypothetical miRNA regulation of a transcription factor or its targets. Corresponding expected enrichment/ depletion of the transcription factor ChIP targets in miRNA-induced upregulated or downregulated transcript sets are displayed in a heat map. A key of color coding representing relative enrichment is given in b. (b) A heat map showing enrichment of the ChIP targets among the different sets of miRNA-regulated transcripts on the horizontal axis. Vertical axis represents the different ChIP data sets with first author and factor that was immunoprecipitated.

Figure 4

Let-7c and miR-294 regulate Lin28, Sall4, cMyc, and nMyc. (a) qRT-PCR for Lin28, Sall4, nMyc, and cMyc 12 hours after transfection with let-7c, miR-294, or a combination of the two. n = 3. (b) Representative Western blot analysis 48 hours after transfection with miRNAs. Quantitation shown in Fig. S10 n = 3. (c) Luciferase analysis of Sall4 and nMyc 3′UTRs. Seed matches for let-7c in the 3′UTRs along with different mutant constructs are diagrammatically represented in the left panel. Luciferase results after co-transfection with let-7c mimic releative to mock transfected are shown in the right panel. All data are represented as mean +/- standard deviation. * indicates p < 0.05 by Bonferroni corrected t-test.

Figure 5

Inhibition of let-7 miRNAs promotes reprogramming to induced pluripotency (a) Fold increase of Oct4∷GFP positive colonies in reprogramming with transduction of 3TFs (Pou5f1/Oct4, Sox2, and Klf4) or 4TFs (+ cMyc) after mock, let-7 inhibitor, or control inhibitor transfection. P-values are indicated for p < 0.01 calculated by Bonferroni corrected t-test. n = 10 for mock and let-7 inhibitor samples and n = 6 for control inhibitor samples (b) A model of the antagonism between the miR-294 and let-7c in the stabilization of the self-renewing and differentiated states. Bold and enlarged genes and arrows are active in the indicated state. Mechanisms of ESCC upregulation of Lin28 and cMyc are unknown and represented by a question mark.