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Oct4/Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells - PubMed

Oct4/Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells

Deborah A Greer Card et al. Mol Cell Biol. 2008 Oct.

Abstract

Oct4 and Sox2 are transcription factors required for pluripotency during early embryogenesis and for the maintenance of embryonic stem cell (ESC) identity. Functional mechanisms contributing to pluripotency are expected to be associated with genes transcriptionally activated by these factors. Here, we show that Oct4 and Sox2 bind to a conserved promoter region of miR-302, a cluster of eight microRNAs expressed specifically in ESCs and pluripotent cells. The expression of miR-302a is dependent on Oct4/Sox2 in human ESCs (hESCs), and miR-302a is expressed at the same developmental stages and in the same tissues as Oct4 during embryogenesis. miR-302a is predicted to target many cell cycle regulators, and the expression of miR-302a in primary and transformed cell lines promotes an increase in S-phase and a decrease in G(1)-phase cells, reminiscent of an ESC-like cell cycle profile. Correspondingly, the inhibition of miR-302 causes hESCs to accumulate in G(1) phase. Moreover, we show that miR-302a represses the productive translation of an important G(1) regulator, cyclin D1, in hESCs. The transcriptional activation of miR-302 and the translational repression of its targets, such as cyclin D1, may provide a link between Oct4/Sox2 and cell cycle regulation in pluripotent cells.

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Figures

FIG. 1.
FIG. 1.

Oct4, Sox2, and Nanog bind to the promoter region of the miR-302 cluster of miRNAs in hESCs. (A) Alignment of the homologous promoter regions of the miR-302 cluster of miRNAs from human, mouse, and chicken. Predicted Oct4, Sox2, and Nanog, binding sites are highlighted in blue, red, and green, respectively. Asterisks indicate sequence homology. (B) Sequences of the double-stranded DNA probe containing the predicted WT Oct4 binding domain and the mutant 1, mutant 2, and double-mutant oligonucleotides (m1, m2, and D) used in EMSA analysis. (C) Sequence of the double-stranded DNA probe containing the predicted Sox2 binding site that is closer to the Oct4 binding sites (−471). m, mutant. (D and E) EMSA analysis performed with extracts of BG-01 cells with the Oct4 binding site. (D) EMSA with 50-fold cold competitors of WT, mutant 1, mutant 2, and double-mutant (m1, m2, and D) oligonucleotides. (E) Supershift with Oct4 antibody. (F and G) EMSA analysis performed with the Sox2 binding site. (H) Oct4, Sox2, and Nanog bind to the promoter region of miR-302 in pluripotent cells. hESCs were cross-linked with formaldehyde, and chromatin was prepared by sheering DNA by sonication. The fragmented chromatin was then immunoprecipitated with antibodies directed toward Oct4, Sox2, Nanog, and nonspecific antibodies (IgG). DNA associated with each of the chromatin immunoprecipitates was amplified by qRT-PCR with primers specific for the promoter regions of miR-302 and Oct4 or GAPDH cDNA as a negative control. The qRT-PCR results shown here are representative examples of the results of at least three experiments performed with the BG-01 cell line. Error bars represent standard deviations. The two major complexes of nuclear extract-bound probe, designated A and B, are indicated with arrows to the right of the gels. +, present; −, absent; FP, free probe; SS, supershift; Comp, competitor; NE, nuclear extract; Ab, antibody; ChIP, chromatin immuniprecipitation.

FIG. 2.
FIG. 2.

Oct4 expression correlates with expression of miR-302 miRNAs during RA treatment. (A, B, and C) hESCs exposed to 1 μM RA for 0 to 10 days were harvested, and RNA and protein extracted from whole-cell lysates were analyzed by qRT-PCR with primers specific to miR-302 transcript, miR-302a, and Oct4 (A); by Western blotting with antibodies specific to Oct4 and α-tubulin (Tub) (B); and by qRT-PCR with primers specific to miR-302b, -c, and -d (C). (D) NTERA2 cells exposed to RA for 10 days were analyzed as described for panel A with primers specific to the miR-302 transcript and miR-302a or by Western blotting with antibodies specific to Oct4 and β-actin. The qRT-PCR and Western blots shown here are representative examples of the results of three experiments performed with H1 cells or NTERA2 cells. +, treated with RA; −, not treated with RA. Error bars represent standard deviations.

FIG. 3.
FIG. 3.

Oct4 and Sox2 transcription factors are required for expression of miR-302 miRNAs. (A) BG-01 cells were transfected with 200 nM nonspecific control (NS) or 100 nM each of Oct4 and Sox2 (O/S) siRNA oligonucleotides as described in Materials and Methods, and whole-cell extracts were made. Oct4, Sox2, and β-actin protein expression levels were analyzed by Western blotting with specific antibodies. (B) BG-01 cells were transfected with pGL3-Basic or pGL3-miR-302 with either nonspecific control siRNA (NS) or Oct4/Sox2 siRNA (O/S) as described for panel A for 48 h. In addition, all samples were transfected with pRL-CMV as a transfection control. Cell extracts were then harvested and analyzed for relative luciferase activity. A schematic of the cloned promoter region in pGL3-miR-302 is shown. luc/ren, luciferase/Renilla activities. (C) BG-01 cells were transfected with 100 nM of nonspecific control (NS), Oct4 siRNA (O), Sox2 siRNA (S), or Oct4/Sox2 siRNA (O/S) as described for panel A, and Oct4, Sox2, and β-actin protein expression levels were analyzed by Western blotting with specific antibodies. (D) qRT-PCR was used to analyze the relative expression levels, normalized to 5S rRNA levels, of the mature miR-302a miRNA in the siRNA-depleted cells. (E) HeLa cells were transfected with pGL3-Basic or pGL3-miR-302 with pCAG-Myc-Oct4 and pCAG-HA-Sox2 cotransfection or empty vector cotransfection. Western blots were probed with Oct4, Sox2, and β-actin antibodies. +, present; −, absent. (F) HeLa cell lysates were analyzed for relative luciferase activities; data are expressed in relative units.

FIG. 4.
FIG. 4.

miR-302 is expressed in early embryogenesis. (A) CD1 embryos were isolated at E3.5, E6.5, E7.5, and E8.5 points of development. RNA was extracted and analyzed by qRT-PCR for levels of Oct4 mRNA, miR-302 transcript, and miR-302a, and the average relative expression levels are shown. (B) E6.5 and E7.5 embryos were analyzed by in situ hybridization with probes for miR-302a (miR-302) and a negative control, as indicated. Top, E6.5 and E7.5 embryos in the same field; bottom, E6.5 and E7.5 embryos, as indicated. Bars show sizes as indicated.

FIG. 5.
FIG. 5.

The expression of miR-302 cluster miRNAs is associated with a short G1 phase. HeLa cells were transfected with pre-miR-302a or negative (neg) control pre-miR. Cell cycle analysis was performed by FACS. Percentages of cells in G1, S, and G2/M are depicted in the graph. Shown here are a typical FACs analysis plot (A) and a graph representing the means ± SEM of the results of three independent experiments (B). (C and D) NHFs were transfected with pre-miR-302a or negative control pre-miR. Shown here are a typical FACs analysis plot (C) and a graph representing the means ± SEM of the results of four independent experiments (D). (E and F) BG-01 cells grown on Matrigel plates were transfected by nucleofection with 200 pmol each miR-302a, -b, -c, and -d inhibitors (inh) or the negative control, as described in Materials and Methods, and cell cycle analysis was performed by FACS. Percentages of cells in G1, S, and G2/M are depicted in the graph. Shown here are a typical FACs analysis plot (E) and a graph representing the means ± SEM of the results of three independent experiments (F).

FIG. 6.
FIG. 6.

Expression analysis of the potential miR-302 target cyclin D1. (A) Western blot analysis of cyclin D1 protein expression and qRT-PCR analysis of cyclin D1 mRNA in extracts of H1 cells treated with RA as described in the Fig. 2 legend. αTub, α-tubulin. (B) Western blot and qRT-PCR analysis of cyclin D1 expression in NTERA2 cells. +, treated with RA; −, not treated with RA. (C) Western blot analysis of Cdk2, Cdk4, Cdk6, and E2F1 protein expression in extracts of H1 cells treated with RA.

FIG. 7.
FIG. 7.

miR-302a binds to the 3′UTR of cyclin D1 to posttranscriptionally repress protein expression. (A) BG-01 cells grown on Matrigel plates were transfected by nucleofection with 200 pmol each of miR-302a, -b, -c, and -d inhibitors (inh) or the negative control (neg), as described in Materials and Methods, and protein and RNA were isolated from whole-cell extracts. qRT-PCR analysis of mature miR-302a levels upon transfection with anti-miR-302 inhibitors was performed. Cyclin D1 and β-actin protein expression levels were analyzed by Western blotting with specific antibodies. RNA isolated from the same cell lysates was analyzed by qRT-PCR with primers specific for cyclin D1 mRNA, and the results were plotted as relative units. (B) Twenty nanomolar pre-miR-302a or negative control pre-miR (neg) was transfected into HeLa cells in duplicate, as indicated. Results of qRT-PCR analysis of mature miR-302a levels upon transfection are shown. The endogenous miRNA miR-24 was also analyzed by qRT-PCR. Protein isolated from whole-cell extracts was analyzed by Western blotting with antibodies directed toward cyclin D1 or α-tubulin (αTub). RNA isolated from the same cell lysates was analyzed by qRT-PCR with primers specific for cyclin D1 mRNA, and the results were plotted as relative units. (C) Schematic of miR-302a, -b, -c, and -d binding site in cyclin D1 3′UTR inserted into the pMIR-REPORT luciferase reporter vector. A section of the cyclin D1 3′UTR containing the predicted miR-302a, -b, -c, and -d binding site (highlighted in red) was inserted downstream of luciferase in the pMIR-REPORT vector. Predicted pairing regions within the WT cyclin D1 3′UTR are noted in red, while those residues altered in the cyclin D1 mutant construct are bolded and italicized. (D) HeLa cells were transfected with the WT cyclin D1 or mutant cyclin D1 vectors, as indicated, with either pre-miR-302a or negative control pre-miR (neg). Additionally, all samples were transfected with pRL-CMV. Cell extracts were then harvested and analyzed for relative luciferase activities. Shown here are the means ± SEM of the results of three independent experiments. (E) BG-01 cells were transfected with the luciferase vectors as described for panel D. Additionally, 25 nM each of miR-302a, -b, -c, and -d inhibitor (inh) or 100 nM anti-miR negative control (neg) was added, as described in Materials and Methods. Relative luciferase activities were then analyzed. Shown here are the means ± SEM of the results of three independent experiments. An asterisk indicates a P value of <0.05. Luc/Ren, luciferase/Renilla activities; D1, cyclin D1; mut, mutant.

FIG. 8.
FIG. 8.

Expression analysis of the potential miR-302 target Cdk4. (A) Western blot analysis of Cdk4 protein expression in BG-01 cells transfected with negative control (neg) or miR-302a, -b, -c, and -d inhibitors (inh) as described for Fig. 7A. (B) Western blot analysis of Cdk4 protein expression in HeLa cells transfected with negative control (neg) or pre-miR-302a as described for Fig. 7B. αTub, α-tubulin.

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