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Myc-regulated microRNAs attenuate embryonic stem cell differentiation.

Lin CH, Jackson AL, Guo J, Linsley PS, Eisenman RN - EMBO J. (2009)

Bottom Line: We further show that the introduction of c-Myc-induced miRNAs into murine ES cells significantly attenuates the downregulation of pluripotency markers on induction of differentiation after withdrawal of the ES cell maintenance factor LIF.In contrast, knockdown of the endogenous miRNAs accelerate differentiation.Our data show that in ES cells c-Myc acts, in part, through a subset of miRNAs to attenuate differentiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-4417, USA.

ABSTRACT
Myc proteins are known to have an important function in stem cell maintenance. As Myc has been shown earlier to regulate microRNAs (miRNAs) involved in proliferation, we sought to determine whether c-Myc also affects embryonic stem (ES) cell maintenance and differentiation through miRNAs. Using a quantitative primer-extension PCR assay we identified miRNAs, including, miR-141, miR-200, and miR-429 whose expression is regulated by c-Myc in ES cells, but not in the differentiated and tumourigenic derivatives of ES cells. Chromatin immunoprecipitation analyses indicate that in ES cells c-Myc binds proximal to genomic regions encoding the induced miRNAs. We used expression profiling and seed homology to identify genes specifically downregulated both by these miRNAs and by c-Myc. We further show that the introduction of c-Myc-induced miRNAs into murine ES cells significantly attenuates the downregulation of pluripotency markers on induction of differentiation after withdrawal of the ES cell maintenance factor LIF. In contrast, knockdown of the endogenous miRNAs accelerate differentiation. Our data show that in ES cells c-Myc acts, in part, through a subset of miRNAs to attenuate differentiation.

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Target gene specificity of Myc-induced miRNAs. (A) The transcript levels of the indicated microRNA targets were validated by real-time PCR. RNA extracted 24 h after transfection of miRNA duplexes into ES cells was used to determine expression of target genes by real-time PCR. Each bar represents the average ΔΔCt on a log2 scale of triplicate sets of experiments after normalization to internal controls (ΔCt) and mock control (ΔΔCt). Error bars show standard error of the mean. (B) Total protein extracted from ES cells 48 h post transfection of miRNA duplexes was analysed by immunoblotting with commercially available antibodies. Mock-transfected cells were used as controls and γ-tubulin as loading control. (C) microRNA target specificity determined by dual luciferase reporter assay. Sequences complementary to the seed region of miR-141, miR-200, and miR-429 were present in the 3′ UTRs of Cdh11, Nrp1, and zfh1, respectively. To disrupt base pairing with miRNAs we mutated the 3′ UTR complementary seed sequences from ‘AACACT' to ‘ACAACT' for the 3′ UTR of Cdh11, from ‘AACAC' to ‘AGCGC' for Nrp1, and from ‘TAATAC' to ‘TCGTAC' for Zfh1a. The wild type or mutant 3′ UTRs derived from the indicated target genes were linked to luciferase and transfected into HeLa cells together with the indicated miRNAs. The fold change represents the ratio of firefly luciferase (FL)/Renilla luciferase (RL). Error bars represent standard error from four independent experiments. **P-value=0.002–0.008; *P-value=0.03.
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f5: Target gene specificity of Myc-induced miRNAs. (A) The transcript levels of the indicated microRNA targets were validated by real-time PCR. RNA extracted 24 h after transfection of miRNA duplexes into ES cells was used to determine expression of target genes by real-time PCR. Each bar represents the average ΔΔCt on a log2 scale of triplicate sets of experiments after normalization to internal controls (ΔCt) and mock control (ΔΔCt). Error bars show standard error of the mean. (B) Total protein extracted from ES cells 48 h post transfection of miRNA duplexes was analysed by immunoblotting with commercially available antibodies. Mock-transfected cells were used as controls and γ-tubulin as loading control. (C) microRNA target specificity determined by dual luciferase reporter assay. Sequences complementary to the seed region of miR-141, miR-200, and miR-429 were present in the 3′ UTRs of Cdh11, Nrp1, and zfh1, respectively. To disrupt base pairing with miRNAs we mutated the 3′ UTR complementary seed sequences from ‘AACACT' to ‘ACAACT' for the 3′ UTR of Cdh11, from ‘AACAC' to ‘AGCGC' for Nrp1, and from ‘TAATAC' to ‘TCGTAC' for Zfh1a. The wild type or mutant 3′ UTRs derived from the indicated target genes were linked to luciferase and transfected into HeLa cells together with the indicated miRNAs. The fold change represents the ratio of firefly luciferase (FL)/Renilla luciferase (RL). Error bars represent standard error from four independent experiments. **P-value=0.002–0.008; *P-value=0.03.

Mentions: Among the genes significantly repressed by both c-Myc and the c-Myc-induced miRNAs, a subset has been implicated in growth arrest and differentiation of a number of cell types (Supplementary Table S3; see Discussion). To validate the expression profiling results, we employed qRT–PCR to assess expression of 20 potential targets 24 h after transfection of duplex miRNAs into ES cells (Figure 5A). We confirmed by northern blotting that the levels of introduced miRNAs are comparable to those following induction by c-Myc (see Supplementary Figure S5). Our results show that introduction of these miRNAs into ES cells is associated with decreased transcript levels for the predicted miRNA targets. Furthermore, using immunoblotting we found that the protein levels of several target genes, such as amphiregulin (Areg), Neuropilin 1 (Nrp1), cadherin 11 (Cdh11), and TGFβ receptor 3 (Tgfbr3), were sharply decreased at 48 h after miRNA transfection (Figure 5B).


Myc-regulated microRNAs attenuate embryonic stem cell differentiation.

Lin CH, Jackson AL, Guo J, Linsley PS, Eisenman RN - EMBO J. (2009)

Target gene specificity of Myc-induced miRNAs. (A) The transcript levels of the indicated microRNA targets were validated by real-time PCR. RNA extracted 24 h after transfection of miRNA duplexes into ES cells was used to determine expression of target genes by real-time PCR. Each bar represents the average ΔΔCt on a log2 scale of triplicate sets of experiments after normalization to internal controls (ΔCt) and mock control (ΔΔCt). Error bars show standard error of the mean. (B) Total protein extracted from ES cells 48 h post transfection of miRNA duplexes was analysed by immunoblotting with commercially available antibodies. Mock-transfected cells were used as controls and γ-tubulin as loading control. (C) microRNA target specificity determined by dual luciferase reporter assay. Sequences complementary to the seed region of miR-141, miR-200, and miR-429 were present in the 3′ UTRs of Cdh11, Nrp1, and zfh1, respectively. To disrupt base pairing with miRNAs we mutated the 3′ UTR complementary seed sequences from ‘AACACT' to ‘ACAACT' for the 3′ UTR of Cdh11, from ‘AACAC' to ‘AGCGC' for Nrp1, and from ‘TAATAC' to ‘TCGTAC' for Zfh1a. The wild type or mutant 3′ UTRs derived from the indicated target genes were linked to luciferase and transfected into HeLa cells together with the indicated miRNAs. The fold change represents the ratio of firefly luciferase (FL)/Renilla luciferase (RL). Error bars represent standard error from four independent experiments. **P-value=0.002–0.008; *P-value=0.03.
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f5: Target gene specificity of Myc-induced miRNAs. (A) The transcript levels of the indicated microRNA targets were validated by real-time PCR. RNA extracted 24 h after transfection of miRNA duplexes into ES cells was used to determine expression of target genes by real-time PCR. Each bar represents the average ΔΔCt on a log2 scale of triplicate sets of experiments after normalization to internal controls (ΔCt) and mock control (ΔΔCt). Error bars show standard error of the mean. (B) Total protein extracted from ES cells 48 h post transfection of miRNA duplexes was analysed by immunoblotting with commercially available antibodies. Mock-transfected cells were used as controls and γ-tubulin as loading control. (C) microRNA target specificity determined by dual luciferase reporter assay. Sequences complementary to the seed region of miR-141, miR-200, and miR-429 were present in the 3′ UTRs of Cdh11, Nrp1, and zfh1, respectively. To disrupt base pairing with miRNAs we mutated the 3′ UTR complementary seed sequences from ‘AACACT' to ‘ACAACT' for the 3′ UTR of Cdh11, from ‘AACAC' to ‘AGCGC' for Nrp1, and from ‘TAATAC' to ‘TCGTAC' for Zfh1a. The wild type or mutant 3′ UTRs derived from the indicated target genes were linked to luciferase and transfected into HeLa cells together with the indicated miRNAs. The fold change represents the ratio of firefly luciferase (FL)/Renilla luciferase (RL). Error bars represent standard error from four independent experiments. **P-value=0.002–0.008; *P-value=0.03.
Mentions: Among the genes significantly repressed by both c-Myc and the c-Myc-induced miRNAs, a subset has been implicated in growth arrest and differentiation of a number of cell types (Supplementary Table S3; see Discussion). To validate the expression profiling results, we employed qRT–PCR to assess expression of 20 potential targets 24 h after transfection of duplex miRNAs into ES cells (Figure 5A). We confirmed by northern blotting that the levels of introduced miRNAs are comparable to those following induction by c-Myc (see Supplementary Figure S5). Our results show that introduction of these miRNAs into ES cells is associated with decreased transcript levels for the predicted miRNA targets. Furthermore, using immunoblotting we found that the protein levels of several target genes, such as amphiregulin (Areg), Neuropilin 1 (Nrp1), cadherin 11 (Cdh11), and TGFβ receptor 3 (Tgfbr3), were sharply decreased at 48 h after miRNA transfection (Figure 5B).

Bottom Line: We further show that the introduction of c-Myc-induced miRNAs into murine ES cells significantly attenuates the downregulation of pluripotency markers on induction of differentiation after withdrawal of the ES cell maintenance factor LIF.In contrast, knockdown of the endogenous miRNAs accelerate differentiation.Our data show that in ES cells c-Myc acts, in part, through a subset of miRNAs to attenuate differentiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-4417, USA.

ABSTRACT
Myc proteins are known to have an important function in stem cell maintenance. As Myc has been shown earlier to regulate microRNAs (miRNAs) involved in proliferation, we sought to determine whether c-Myc also affects embryonic stem (ES) cell maintenance and differentiation through miRNAs. Using a quantitative primer-extension PCR assay we identified miRNAs, including, miR-141, miR-200, and miR-429 whose expression is regulated by c-Myc in ES cells, but not in the differentiated and tumourigenic derivatives of ES cells. Chromatin immunoprecipitation analyses indicate that in ES cells c-Myc binds proximal to genomic regions encoding the induced miRNAs. We used expression profiling and seed homology to identify genes specifically downregulated both by these miRNAs and by c-Myc. We further show that the introduction of c-Myc-induced miRNAs into murine ES cells significantly attenuates the downregulation of pluripotency markers on induction of differentiation after withdrawal of the ES cell maintenance factor LIF. In contrast, knockdown of the endogenous miRNAs accelerate differentiation. Our data show that in ES cells c-Myc acts, in part, through a subset of miRNAs to attenuate differentiation.

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