Abstract

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.

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Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE30245.

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Acknowledgements

We thank D. Rivera, T. Green, T. Bhimdi, G. Verstappen, C. Surka, S. Silver, A. Brown, D. Lam and O. Ram for technical help; C. Gifford, S. Markoulaki and R. Jaenisch for providing cell lines used in this study; P. Tsang, B. Curry, A. Tsalenko and Agilent Technologies for microarray and technical help; B. Challis and Active Motif for antibodies; G. Geiss, R. Boykin and Nanostring technologies for technical help; E. Wang and C. Burge for help with RNA immunoprecipitation experiments and helpful discussions; P. Gupta, A. Gnirke, J. Cassady, E. Lieberman-Aiden, M. Cabili and M. Thompson for discussions and ideas; and L. Gaffney for assistance with figures. M. Guttman is a Vertex scholar. This work was funded by NHGRI, a Center for Excellence for Genomic Science, the Merkin Foundation for Stem Cell Research, and funds from the Broad Institute of MIT and Harvard.

Author information

Author notes

    • Alexander Meissner
    • , Aviv Regev
    • , John L. Rinn
    •  & David E. Root

    These authors contributed equally to this work.

Affiliations

  1. Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA

    • Mitchell Guttman
    • , Julie Donaghey
    • , Manuel Garber
    • , Jennifer K. Grenier
    • , Glen Munson
    • , Geneva Young
    • , Xiaoping Yang
    • , Ido Amit
    • , Alexander Meissner
    • , Aviv Regev
    • , John L. Rinn
    • , David E. Root
    •  & Eric S. Lander
  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Mitchell Guttman
    • , Bryce W. Carey
    • , Aviv Regev
    •  & Eric S. Lander
  3. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA

    • Bryce W. Carey
  4. Genomics Research and Development, Agilent Technologies, Santa Clara, California 95051, USA

    • Anne Bergstrom Lucas
    • , Robert Ach
    •  & Laurakay Bruhn
  5. Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Alexander Meissner
    •  & John L. Rinn
  6. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02114, USA

    • Eric S. Lander

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Contributions

M. Guttman and E.S.L. conceived and designed the overall project with help from A.M., A.R., J.L.R. and D.E.R.; M. Guttman and J.D. designed experiments with help from J.K.G., X.Y. (RNAi), B.W.C. (pluripotency assays) and I.A. (RNA IP); M. Guttman, J.D., G.M., A.B.L., R.A. and G.Y. performed experiments; M. Guttman, J.D. and M. Garber analysed data; L.B., A.M. and D.E.R. provided reagents; and M. Guttman and E.S.L. wrote the manuscript.

Competing interests

A.B.L., R.A. and L.B. are employees of and own stock in Agilent technologies.

Corresponding authors

Correspondence to Mitchell Guttman or Eric S. Lander.

Supplementary information

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  1. 1.

    Supplementary Figures

    The file contains Supplementary Figures 1-17 with legends.

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  1. 1.

    Supplementary Tables

    This file contains Supplementary Tables 1–19 as follows: Table 1 ESC lincRNA names, genomic coordinates, sequences, and primers; Table 2 shRNA sequences and on-target knockdown levels; Table 3 Affected genes upon knockdown of lincRNA and protein-coding genes; Table 4 Number of genes affected upon knockdown; Table 5 Overlapping gene expression affects for second hairpins; Table 6 Distance to closest affected protein-coding gene, Table 7 Nanog-Luciferase levels and Alamar levels after infection; Table 8 mRNA expression levels of pluripotency markers after knockdown of lincRNA and protein-coding genes; Table 9 Primer sequences used for pluripotency and differentiation markers; Table 10 Oct4 expression levels across second best hairpins; Table 11 lincRNA knockdown projections; Table 12 Lineage expression profiles; Table 13 Lineage expression expression changes upon transcription factor knockdown, Table 16 incRNA expression changes upon retinoic acid induced differentiation Table17 lincRNA associations with chromatin proteins; Table 18 Tested chromatin complexes and antibodies used and Table19 Overlap in expression between chromatin proteins and lincRNAs.

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https://doi.org/10.1038/nature10398

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