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Dicer-microRNA-Myc circuit promotes transcription of hundreds of long noncoding RNAs

Nature Structural & Molecular Biology volume 21, pages 585–590 (2014)Cite this article

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Abstract

Long noncoding RNAs (lncRNAs) are important regulators of cell fate, yet little is known about mechanisms controlling lncRNA expression. Here we show that transcription is quantitatively different for lncRNAs and mRNAs—as revealed by deficiency of Dicer (Dcr), a key RNase that generates microRNAs (miRNAs). Dcr loss in mouse embryonic stem cells led unexpectedly to decreased levels of hundreds of lncRNAs. The canonical Dgcr8-Dcr-miRNA pathway is required for robust lncRNA transcriptional initiation and elongation. Computational and genetic epistasis analyses demonstrated that Dcr activation of the oncogenic transcription factor cMyc is partly responsible for lncRNA expression. A quantitative metric of mRNA-lncRNA decoupling revealed that Dcr and cMyc differentially regulate lncRNAs versus mRNAs in diverse cell types and in vivo. Thus, numerous lncRNAs may be modulated as a class in development and disease, notably where Dcr and cMyc act.

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Figure 1: Global lncRNA downregulation in Dcr-KO mESCs.
Figure 2: Canonical microRNA pathway and miR-295 are required for lncRNA expression.
Figure 3: Dicer promotes lncRNA expression at the level of lncRNA transcription.
Figure 4: cMyc-binding sites of lncRNAs affect their sensitivity to Dcr loss.
Figure 5: Loss of Dcr and cMyc leads to a more severe change in expression of lncRNAs than of mRNAs in diverse cellular contexts.

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Acknowledgements

We thank members of the Chang laboratory, and P. Sharp (Massachusetts Institute of Technology) and A. Giraldez (Yale) for discussion, and for sharing unpublished data. We thank R. Blelloch (University of California, San Francisco) and A. Bradley (Wellcome Trust Sanger Institute) for sharing DGCR8 WT and KO mESCs, and cMyc KO mESCs respectively. G.X.Y.Z. was supported by the Leukemia and Lymphoma Society (grant 5549-13 to G.X.Y.Z.) and a Dean's Fellowship from Stanford University. The study was supported by the US National Institutes of Health (grant R01-CA118750 to H.Y.C.) and California Institute for Regenerative Medicine (grant RB4-05763 to H.Y.C.). H.Y.C. is supported as an Early Career Scientist of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA

    Grace X Y Zheng, Brian T Do, Dan E Webster, Paul A Khavari & Howard Y Chang

  2. Howard Hughes Medical Institute, Stanford, California, USA

    Howard Y Chang

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  1. Grace X Y Zheng
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Contributions

G.X.Y.Z. and H.Y.C. initiated the project. G.X.Y.Z. and H.Y.C. designed the experiments. G.X.Y.Z. performed the experiments and the computational analysis. B.T.D., D.E.W. and P.A.K. designed and implemented bioinformatics and microarray screens. The manuscript was prepared by G.X.Y.Z. and H.Y.C. with input from all authors.

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Correspondence to Howard Y Chang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of sequencing data.

Sequencing tracks of (a) Casp2, (b) cMyc, (c) lncRNA NR_015519, and (d) lnc543. The y-axis represents number of reads per million. Transcript structure is shown at the bottom in black, and exons are shown in black boxes. (e) Cdfs of LFC of lncRNAs (red) and coding genes (blue) between Dcr WT and KO mESCs. The expression of coding genes in WT mESCs is matched to that of lncRNAs. lncRNAs are significantly downregulated in Dcr KO mESCs relative to coding genes (p<2.2x10-16). (f) Cdfs of LFC of mRNA expression between Dcr WT and KO mESCs. Plots include “predicted coding targets” of miR-295 (blue line), and “coding control” (grey line). “predicted coding targets” include mRNAs that have a miR-295 target in their 3' UTRs. The “coding control” mRNA set was selected to match the predicted targets in expression, 3' UTR length and composition. “predicted coding targets” are derepressed in Dcr KO mESCs (p≤ 2.2x10−16 by rank sum test). (g) Cdfs of LFC of coding targets of miR-295, using the full-length sequence. Similarly, “predicted coding targets” are depressed in Dcr KO mESCs (p = 1.33x10−10). (h) Cdfs of LFC in lncRNA expression between Dcr WT and KO mESCs. Plots include “predicted lncRNA targets” of miR-295 (red line), and “lncRNA control” (grey line). “predicted lncRNA targets” include lncRNAs that have a miR-295 target in their full length sequences. “predicted lncRNA targets” are derepressed in Dcr KO mESCs (p = 1.66x10−3).

Supplementary Figure 2 lncRNAs are regulated by DGCR8, DCR and miR-295.

(a) Validation of Nanostring data against the RNA-seq data. X-axis, LFC of lncRNAs in Dcr KO and WT mESCs calculated from the RNA-seq data. Y-axis, LFC of lncRNAs detected by nanostring. r = 0.75. (b) Overexpression of Dcr and miR-295 can rescue the expression of miR-295 in Dcr KO mESCs. RT-qPCR of miR-295 after transfection of Dcr, Dcr Mutant (DcrMut), miR-295 or control mimics (ctl). (c) Overexpression of miR-295 can rescue the expression of miR-295 in Dgcr8 KO mESCs. RT-qPCR of miR-295 after transfection of miR-295 or control mimics (ctl) in Dgcr8 KO mESCs. (d) miR-295 and Dcr can rescue the expression of down-regulated lncRNAs. RT-qPCR of lncRNAs after miR-295 overexpression (blue) and Dcr overexpression (pink). Their LFC between Dcr KO and WT cells is plotted (in green) as a comparison. (e) miR-96 cannot rescue the expression of down-regulated lncRNAs. RT-qPCR of lncRNAs after miR-295 overexpression (blue) and miR-96 overexpression (grey). (f) miR-295 can rescue the expression of downregulated lncRNAs in DGCR8 KO mESCs. RT-qPCR of lncRNAs after miR-295 overexpression in DGCR8 KO cells (blue). Their LFC between DGCR8 KO and WT cells is plotted (in green) as a comparison. (g) Scatter plot of LFC of lncRNAs in Dcr KO and Dgcr8 KO cells measured by Nanostring. X-axis, LFC of lncRNAs in Dcr KO and WT mESCs. Y-axis, LFC of lncRNAs in Dgcr8 KO and WT mESCs. r = 0.72. For b-f, n≥3, mean ± S.E.M.

Supplementary Figure 3 Validation of half-life and chromatin data.

(a) Half life of 7 mRNAs was compared to published half life data. The correlation is 0.83. (b) Synthesis rate of 7 mRNAs as positive controls. (c) Half life of 7 mRNAs as positive controls. For a-c, n≥3, mean ± S.E.M. (d) Down-regulated coding genes have a similar H3K4me3 signal in WT and Dcr KO cells. Average signal intensity of H3K4me3 across down-regulated mRNA genes in WT (magenta) and Dcr KO (green) mESCs. Y-axis represents average log2 value of normalized reads. (e) Average signal intensity of H3K36me3 across down-regulated mRNA genes in WT (magenta) and Dcr KO (green) mESCs. Y-axis represents average log2 value of normalized reads.

Supplementary Figure 4 cMyc has an important role in lncRNA expression of mESCs.

(a) Relative expression change (RPKM) of candidate factors between Dcr KO and WT mESCs. (b) Heatmap of cMyc ChIP-seq signals at down-regulated and up-regulated lncRNAs. (c) Cdfs of LFC in lncRNA expression between Dcr WT and KO mESCs are plotted. Plots include “coding genes” (blue line), “lncRNAs” (dark red line), and “lncRNAs with cMyc binding sites (red line). lncRNAs with cMyc binding sites are much more down-regulated than the rest of lncRNAs (p ≤ 2.2x10−16 by rank sum test). (d) Cdfs of LFC in mRNA expression between Dcr WT and KO mESCs are plotted. Plots include “mRNAs with cMyc binding sites” (dark blue line) and “coding genes without cMyc binding sites” (black line). (e) Average signal intensity of H3K4me3 across mRNA genes with (top) and without (bottom) cMyc binding sites in WT (magenta) and Dcr KO (green) mESCs. (f) Average signal intensity of H3K36me3 across mRNA genes with (top) and without (bottom) cMyc binding sites in WT (magenta) and Dcr KO (green) mESCs. Y-axis represents average log2 value of normalized reads. (g) Average signal intensity of Pol2 occupancy across lncRNAs with cMyc binding sites (top) and lncRNAs without cMyc binding sites (bottom) before (black) and after (green) the treatment with cMyc inhibitor. Y-axis represents average value of rank normalized reads.

Supplementary Figure 5 Genomic organization of lncRNAs affects their sensitivity to Dcr loss.

(a) Sequencing track of a divergent lncRNA (red)-coding gene (blue) pair expression in WT (magenta) and Dcr KO (green) mESCs. (b) Classification of 2229 lncRNAs based on their orientation and distances to neighboring coding genes. (c) Cdfs of divergent lncRNAs (black) and their corresponding coding gene pair (dark blue). (d) lncRNAs that are divergent from coding genes tend to be downregulated in Dcr KO mESCs. There are 41% of them relative to 14% of them in up-regulated lncRnAs. (e) Cdfs of LFC in lncRNA expression between Dcr WT and KO mESCs are plotted. “cMyc-only lncRNAs” (red) are lncRNAs that only have cMyc binding sites. “divergent-only lncRNAs” (black) are lncRNAs that are only divergent from coding genes. “cMyc + divergent” (purple) lncRNAs are lncRNAs that have both cMyc binding sites and are divergent from coding genes. All 3 classes of lncRNAs are significantly more downregulated (p < 2.2x10−16) in Dcr KO mESCs relative to “all lncRNAs” (dark red), although they are not significantly different from each other.

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Zheng, G., Do, B., Webster, D. et al. Dicer-microRNA-Myc circuit promotes transcription of hundreds of long noncoding RNAs. Nat Struct Mol Biol 21, 585–590 (2014). https://doi.org/10.1038/nsmb.2842

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