c-Myc-regulated microRNAs modulate E2F1 expression

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Abstract

MicroRNAs (miRNAs) are 21–23 nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs1. miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation and apoptosis2. Although strict tissue- and developmental-stage-specific expression is critical for appropriate miRNA function, mammalian transcription factors that regulate miRNAs have not yet been identified. The proto-oncogene c-MYC encodes a transcription factor that regulates cell proliferation, growth and apoptosis3. Dysregulated expression or function of c-Myc is one of the most common abnormalities in human malignancy4. Here we show that c-Myc activates expression of a cluster of six miRNAs on human chromosome 13. Chromatin immunoprecipation experiments show that c-Myc binds directly to this locus. The transcription factor E2F1 is an additional target of c-Myc that promotes cell cycle progression5,6,7. We find that expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. These findings expand the known classes of transcripts within the c-Myc target gene network, and reveal a mechanism through which c-Myc simultaneously activates E2F1 transcription and limits its translation, allowing a tightly controlled proliferative signal.

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Figure 1: MicroRNA expression profiling of P493-6 cells with high and low c-Myc expression.
Figure 2: c-Myc induces expression of the miR-17 cluster.
Figure 3: c-Myc binds directly to the mir-17 cluster genomic locus.
Figure 4: miR-17-5p and miR-20a regulate E2F1 translational yield.

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Acknowledgements

The authors thank D. Eick and J. Sedivy for cell lines, J. Kim for assistance with ChIP, and F. Spencer and M. Awad for critical reading of the manuscript. This work was supported in part by a grant from the NIH (C.V.D.) and a Sidney Kimmel Pilot Project Grant (J.T.M.). C.V.D. is the Johns Hopkins Family Professor in Oncology Research, and J.T.M. is a March of Dimes Basil O'Connor Scholar.

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Correspondence to Joshua T. Mendell.

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Supplementary information

Supplementary Figure S1

Conservation of the miRNA binding sites in the 3' UTR of E2F1 mRNA and reporter assays demonstrating direct regulation of this transcript by miR-17-5p and miR-20a. (PDF 86 kb)

Supplementary Figure S2

Northern and western blot analysis of E2F1 and c-Myc mRNA and protein levels during a serum-stimulation time-course of primary human fibroblasts. (PDF 486 kb)

Supplementary Figure Legends

Legends to accompany the above Supplementary Figures. (DOC 21 kb)

Supplementary Table S1

Sequences of probes spotted on miRNA array. (XLS 147 kb)

Supplementary Table S2

Sequences of primers used in ChIP analysis of the miR-17 cluster. (XLS 19 kb)

Supplementary Methods

Additional information on methods used in this study, including the construction of E2F1 and PTEN luciferase reporter plasmids and the luciferase assays. (DOC 21 kb)

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O'Donnell, K., Wentzel, E., Zeller, K. et al. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435, 839–843 (2005) doi:10.1038/nature03677

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