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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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miRNAs regulated by c-Myc in murine ES cells. (A) Scheme showing the experimental design for identification of miRNAs regulated by c-Myc in ES cells. c-Myc level was increased by lentiviral-delivered overexpression of c-Myc in ES cells (see Supplementary Figure S1). These ES cells, their differentiated haematopoietic stem/progenitor cell derivatives (HSP Myc+) and tumours after injection of HSP c-Myc+ cells into irradiated mice, were subjected to miRNA profiling. (B) Heatmap of miRNAs displaying significant regulation by c-Myc in ES cells and ES cell-derived differentiated cells or tumours. HSP: haematopoietic stem/progenitor cells derived by differentiation of ES cells; Tumours1–4: mixed T- and B-cell tumours derived by transplantation of Myc+ HSPs into irradiated mice. Note that results are shown for two HSP, and four tumour cell isolates independently derived from the same ES cell line. Bottom row shows miRNAs levels after c-Myc knockdown in WT ES cells. Inset: Fold change in miRNA copy number represents the ratio of miRNA level in ES cell in which c-Myc has been introduced (Myc+), or knocked down by c-myc shRNA (ES Myc KD), relative to lentiviral vector (see Supplementary Figure S1 for quantitation). Grey boxes represent no detectable signal.
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f1: miRNAs regulated by c-Myc in murine ES cells. (A) Scheme showing the experimental design for identification of miRNAs regulated by c-Myc in ES cells. c-Myc level was increased by lentiviral-delivered overexpression of c-Myc in ES cells (see Supplementary Figure S1). These ES cells, their differentiated haematopoietic stem/progenitor cell derivatives (HSP Myc+) and tumours after injection of HSP c-Myc+ cells into irradiated mice, were subjected to miRNA profiling. (B) Heatmap of miRNAs displaying significant regulation by c-Myc in ES cells and ES cell-derived differentiated cells or tumours. HSP: haematopoietic stem/progenitor cells derived by differentiation of ES cells; Tumours1–4: mixed T- and B-cell tumours derived by transplantation of Myc+ HSPs into irradiated mice. Note that results are shown for two HSP, and four tumour cell isolates independently derived from the same ES cell line. Bottom row shows miRNAs levels after c-Myc knockdown in WT ES cells. Inset: Fold change in miRNA copy number represents the ratio of miRNA level in ES cell in which c-Myc has been introduced (Myc+), or knocked down by c-myc shRNA (ES Myc KD), relative to lentiviral vector (see Supplementary Figure S1 for quantitation). Grey boxes represent no detectable signal.

Mentions: To identify ES-specific c-Myc-induced miRNAs, we analysed three cell populations that permit us to compare ES cells to their differentiated and tumourigenic derivatives: (i) murine AK7 ES cells (ii) induced haematopoietic stem/progenitor cells (HSPs) derived from these ES cells on differentiation, and (iii) tumours from mice transplanted with ES cell-derived HSPs (Figure 1A). To determine the effects of increasing c-Myc levels, we used a c-myc-expressing lentiviral vector in ES cells (hereafter referred to as c-Myc+ cells) and identified c-Myc-induced miRNAs after normalization to a lentiviral vector control. We observed an 8–14-fold increase in c-myc RNA and protein levels as determined by qRT–PCR and immunoblotting, respectively (Supplementary Figure S1). We also generated differentiated cells from control and c-Myc+ ES cells. Treatment of murine ES cells with the cytokines IL-3, IL-6, and SCF resulted in induction of HSPs (Kushida et al, 2001; Burt et al, 2004). Finally, mixed T- and B-cell tumours were collected from irradiated mice transplanted with cytokine-induced c-Myc+ HSPs and compared with vector control transplanted HSPs in the miRNA profiling experiment (Supplementary Figure S2).


Myc-regulated microRNAs attenuate embryonic stem cell differentiation.

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

miRNAs regulated by c-Myc in murine ES cells. (A) Scheme showing the experimental design for identification of miRNAs regulated by c-Myc in ES cells. c-Myc level was increased by lentiviral-delivered overexpression of c-Myc in ES cells (see Supplementary Figure S1). These ES cells, their differentiated haematopoietic stem/progenitor cell derivatives (HSP Myc+) and tumours after injection of HSP c-Myc+ cells into irradiated mice, were subjected to miRNA profiling. (B) Heatmap of miRNAs displaying significant regulation by c-Myc in ES cells and ES cell-derived differentiated cells or tumours. HSP: haematopoietic stem/progenitor cells derived by differentiation of ES cells; Tumours1–4: mixed T- and B-cell tumours derived by transplantation of Myc+ HSPs into irradiated mice. Note that results are shown for two HSP, and four tumour cell isolates independently derived from the same ES cell line. Bottom row shows miRNAs levels after c-Myc knockdown in WT ES cells. Inset: Fold change in miRNA copy number represents the ratio of miRNA level in ES cell in which c-Myc has been introduced (Myc+), or knocked down by c-myc shRNA (ES Myc KD), relative to lentiviral vector (see Supplementary Figure S1 for quantitation). Grey boxes represent no detectable signal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2744176&req=5

f1: miRNAs regulated by c-Myc in murine ES cells. (A) Scheme showing the experimental design for identification of miRNAs regulated by c-Myc in ES cells. c-Myc level was increased by lentiviral-delivered overexpression of c-Myc in ES cells (see Supplementary Figure S1). These ES cells, their differentiated haematopoietic stem/progenitor cell derivatives (HSP Myc+) and tumours after injection of HSP c-Myc+ cells into irradiated mice, were subjected to miRNA profiling. (B) Heatmap of miRNAs displaying significant regulation by c-Myc in ES cells and ES cell-derived differentiated cells or tumours. HSP: haematopoietic stem/progenitor cells derived by differentiation of ES cells; Tumours1–4: mixed T- and B-cell tumours derived by transplantation of Myc+ HSPs into irradiated mice. Note that results are shown for two HSP, and four tumour cell isolates independently derived from the same ES cell line. Bottom row shows miRNAs levels after c-Myc knockdown in WT ES cells. Inset: Fold change in miRNA copy number represents the ratio of miRNA level in ES cell in which c-Myc has been introduced (Myc+), or knocked down by c-myc shRNA (ES Myc KD), relative to lentiviral vector (see Supplementary Figure S1 for quantitation). Grey boxes represent no detectable signal.
Mentions: To identify ES-specific c-Myc-induced miRNAs, we analysed three cell populations that permit us to compare ES cells to their differentiated and tumourigenic derivatives: (i) murine AK7 ES cells (ii) induced haematopoietic stem/progenitor cells (HSPs) derived from these ES cells on differentiation, and (iii) tumours from mice transplanted with ES cell-derived HSPs (Figure 1A). To determine the effects of increasing c-Myc levels, we used a c-myc-expressing lentiviral vector in ES cells (hereafter referred to as c-Myc+ cells) and identified c-Myc-induced miRNAs after normalization to a lentiviral vector control. We observed an 8–14-fold increase in c-myc RNA and protein levels as determined by qRT–PCR and immunoblotting, respectively (Supplementary Figure S1). We also generated differentiated cells from control and c-Myc+ ES cells. Treatment of murine ES cells with the cytokines IL-3, IL-6, and SCF resulted in induction of HSPs (Kushida et al, 2001; Burt et al, 2004). Finally, mixed T- and B-cell tumours were collected from irradiated mice transplanted with cytokine-induced c-Myc+ HSPs and compared with vector control transplanted HSPs in the miRNA profiling experiment (Supplementary Figure S2).

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.

Show MeSH
Related in: MedlinePlus