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. 2011 Oct 15;25(20):2173-86.
doi: 10.1101/gad.17221311.

Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

Inna Lipchina  1 , Yechiel Elkabetz, Markus Hafner, Robert Sheridan, Aleksandra Mihailovic, Thomas Tuschl, Chris Sander, Lorenz Studer, Doron Betel
Affiliations

Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

Inna Lipchina et al. Genes Dev. .

Abstract

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area.

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Figures

Figure 1.
Figure 1.
MicroRNA sequencing profile in human stem cells. MicroRNA levels were measured by deep sequencing in five hESC-derived cell populations: hESCs, R-NSCs, NPCs, EBs, and MPCs. To obtain homogenous cell populations, the cells were sorted by stage-specific markers or mechanically purified. The top panel shows representative images of cell morphology and marker expression at each differentiation stage. In the bottom panel, color grouping indicates microRNAs that are members of the same primary transcript and therefore are cotranscribed. The mir-302 cluster that encodes miR-302a/b/c/d and miR-367 is the most abundant cluster in hESCs and its expression levels rapidly decline upon differentiation. Other abundant microRNAs include mir-17∼92 and its paralog, mir-106a; mir-21; and mir-103, which are persistently expressed in differentiated hESCs (see also Supplemental Table S1; Supplemental Fig. S1).
Figure 2.
Figure 2.
Experimental and computational identification of miR-302/367 targets in hESCs. (A) PAR-CLIP data provide precise location of the AGO2-binding site with hallmark T-to-C mutation at the cross-linked site. This position is typically upstream of the complement microRNA seed sequence shown in the novel SLAIN1 target site identified in this study. The top panel represents an miR-302a predicted target site (at position 145 in the 3′ UTR), and the bottom panel depicts the mapped sequence reads and mismatched positions in green. (B) Summary of the genomic mapping of the CCRs identified by PAR-CLIP. Of the total 7527 CCRs, 4794 (>63%) were mapped to annotated genes. Orange bars indicate the number of CCRs with predicted target sites for one or more of the endogenously expressed (top 29) seed sequences in hESCs, whereas red bars indicate the number of CCRs with no predicted sites for these microRNAs. (C) A summary of the number of CCRs targeted by each of the top 29 hESC-expressed microRNA seed families. Average cross-link is the average fraction of reads in the CCRs with T-to-C mutations (see also Supplemental File S1; Supplemental Table S2). (D) Inhibition of miR-302/367 results in up-regulation of their predicted targets. The cumulative distribution of the log2 expression change of miR-302/367 targets is significantly increased after miR-302/367 inhibition relative to the background set that lacks miR-302/367 predicted targets. Similarly, the miR-302/367 targeted genes identified in the PAR-CLIP experiments are also significantly up-regulated following miR-302/367 inhibition. In contrast, targets of miR-21, another highly expressed microRNA in hESCs, do not undergo a significant change in expression, indicating specific inhibition of miR-302/367. (E) In contrast to inhibition, ectopic expression of miR-302/367 results in only a minor decrease in the expression of their targets.
Figure 3.
Figure 3.
High-confidence microRNA-302/367 targets. (A) The intersection between PAR-CLIP data and the miR-302/367 perturbation experiments identifies the set of most regulated targets. The set of 146 high-confidence miR-302/367 targets is defined as those genes with favorable mirSVR scores (Betel et al. 2010) that are significantly up-regulated after miR-302/367 inhibition and have identified PAR-CLIP sites in their 3′ UTRs. (B) The full list of high-confidence miR-302/367 targets in undifferentiated hESCs. Included in this list are a number of previously identified miR-302 targets (e.g., LEFTY1/2 [Rosa et al. 2009], CDKN1A [Wang et al. 2008], and TGFBR2 [Subramanyam et al. 2011]), many that are implicated in cell cycle regulation (e.g., BTG1,2,3), and negative regulators of BMP and TGF-β signaling.
Figure 4.
Figure 4.
The miR-302/367 cluster promotes pluripotency and modulates BMP signaling during differentiation. (A) FACS-based cell cycle analysis on propidium iodide (PI)-stained cells was used to determine the percentage of cells in a given cell cycle phase. Inhibition of miR-302/367 by antagomirs increased the fraction of cells in G1 phase relative to control (n = 3 independent experiments: antagomir vs. control: P < 0.05 for G1, S, and G2/M comparisons). (B) Inhibition of miR-302/367 by antagomirs results in reduction of pluripotency as measured by FACS analysis of the pluripotency marker SSEA-3. (C) Quantification of hESC clonogenicity as determined by the number of OCT4+ colonies 7 d after replating at clonal density. (D) qRT–PCR for early neural marker PAX6 after 7 d of differentiation in knockout serum replacement (KSR)-based medium containing inhibitors of BMP (Noggin) and TGF-β/Activin/Nodal (SB-431542) signaling pathways. (E) qRT–PCR for trophectoderm markers CDX2, KRT7, and CGA after 3 d of differentiation in RPMI medium with BMP4. (F) qRT–PCR analysis for BMP target gene ID1 (left panel) and luciferase assay of BMP reporter Tlx2-lux (right panel) 36 h after miR-302/367 inhibition by antagomir. (G) qRT–PCR of ID1 48 h after siRNA-mediated knockdown of targeted genes. (H) qRT–PCR of PAX6 after 7 d of differentiation in KSR-based medium containing inhibitor of TGF-β/Activin/Nodal signaling and simultaneous siRNA knockdown of DAZAP2, SLAIN1, and TOB2. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 compared with control antagomir mimic or siRNA (see also Supplemental Figs. S7, S8).
Figure 5.
Figure 5.
miR-302/367 targets modulate TGF-β and BMP signaling. In hESCs, neural induction is initiated by inhibition of TGF-β and BMP signaling. The rapid decline in mir-302 levels upon neural induction suggests that miR-302/367 promotes self-renewal and pluripotency and represses neural differentiation, possibly by suppressing inhibitors of TGF-β and BMP pathways. In this study, we identified three novel miR-302/367 targets that inhibit BMP signaling. Thus, by promoting TGF-β and BMP signaling at the ESC stage through down-regulation of pathway inhibitors, miR-302/367 promotes the pluripotency stage and attenuates neural differentiation.
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