lincRNAs act in the circuitry controlling pluripotency and differentiation


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Functional affects of lincRNAs.
Figure 2: lincRNAs are critical for the maintenance of pluripotency.
Figure 3: lincRNAs repress specific differentiation lineages.
Figure 4: lincRNAs are direct regulatory targets of the ES cell transcriptional circuitry.
Figure 5: lincRNAs physically interact with chromatin regulatory proteins.
Figure 6: A model for lincRNA integration into the molecular circuitry of the cell.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

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


  1. 1

    The FANTOM Consortium The transcriptional landscape of the mammalian genome. Science 309, 1559–1563 (2005)

    ADS  Article  Google Scholar 

  2. 2

    Guttman, M. et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458, 223–227 (2009)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Guttman, M. et al. Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nature Biotechnol. 28, 503–510 (2010)

    CAS  Article  Google Scholar 

  4. 4

    Khalil, A. M. et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl Acad. Sci. USA 106, 11667–11672 (2009)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Ponjavic, J., Ponting, C. P. & Lunter, G. Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs. Genome Res. 17, 556–565 (2007)

    CAS  Article  Google Scholar 

  6. 6

    Mattick, J. S. The genetic signatures of noncoding RNAs. PLoS Genet. 5, e1000459 (2009)

    Article  Google Scholar 

  7. 7

    Koziol, M. J. & Rinn, J. L. RNA traffic control of chromatin complexes. Curr. Opin. Genet. Dev. 20, 142–148 (2010)

    CAS  Article  Google Scholar 

  8. 8

    De Santa, F. et al. A large fraction of extragenic RNA pol II transcription sites overlap enhancers. PLoS Biol. 8, e1000384 (2010)

    Article  Google Scholar 

  9. 9

    Kim, T. K. et al. Widespread transcription at neuronal activity-regulated enhancers. Nature 465, 182–187 (2010)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Ebisuya, M., Yamamoto, T., Nakajima, M. & Nishida, E. Ripples from neighbouring transcription. Nature Cell Biol. 10, 1106–1113 (2008)

    CAS  Article  Google Scholar 

  11. 11

    Ørom, U. A. et al. Long noncoding RNAs with enhancer-like function in human cells. Cell 143, 46–58 (2010)

    Article  Google Scholar 

  12. 12

    Huarte, M. et al. A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell 142, 409–419 (2010)

    CAS  Article  Google Scholar 

  13. 13

    Rinn, J. L. et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311–1323 (2007)

    CAS  Article  Google Scholar 

  14. 14

    Smith, A. G. Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435–462 (2001)

    CAS  Article  Google Scholar 

  15. 15

    Jaenisch, R. & Young, R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132, 567–582 (2008)

    CAS  Article  Google Scholar 

  16. 16

    Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008)

    CAS  Article  Google Scholar 

  17. 17

    Ivanova, N. et al. Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538 (2006)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Boyer, L. A. et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349–353 (2006)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Bernstein, B. E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006)

    CAS  Article  Google Scholar 

  20. 20

    Fazzio, T. G., Huff, J. T. & Panning, B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell 134, 162–174 (2008)

    CAS  Article  Google Scholar 

  21. 21

    Bilodeau, S., Kagey, M. H., Frampton, G. M., Rahl, P. B. & Young, R. A. SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state. Genes Dev. 23, 2484–2489 (2009)

    CAS  Article  Google Scholar 

  22. 22

    Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006)

    CAS  Article  Google Scholar 

  23. 23

    Hu, G. et al. A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes Dev. 23, 837–848 (2009)

    CAS  Article  Google Scholar 

  24. 24

    Plath, K., Mlynarczyk-Evans, S., Nusinow, D. A. & Panning, B. Xist RNA and the mechanism of X chromosome inactivation. Annu. Rev. Genet. 36, 233–278 (2002)

    CAS  Article  Google Scholar 

  25. 25

    Koerner, M. V., Pauler, F. M., Huang, R. & Barlow, D. P. The function of non-coding RNAs in genomic imprinting. Development 136, 1771–1783 (2009)

    CAS  Article  Google Scholar 

  26. 26

    Ponjavic, J., Oliver, P. L., Lunter, G. & Ponting, C. P. Genomic and transcriptional co-localization of protein-coding and long non-coding RNA pairs in the developing brain. PLoS Genet. 5, e1000617 (2009)

    Article  Google Scholar 

  27. 27

    Sproul, D., Gilbert, N. & Bickmore, W. A. The role of chromatin structure in regulating the expression of clustered genes. Nature Rev. Genet. 6, 775–781 (2005)

    CAS  Article  Google Scholar 

  28. 28

    Silva, J. et al. Nanog is the gateway to the pluripotent ground state. Cell 138, 722–737 (2009)

    CAS  Article  Google Scholar 

  29. 29

    Chambers, I. et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003)

    CAS  Article  Google Scholar 

  30. 30

    Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003)

    CAS  Article  Google Scholar 

  31. 31

    Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151–159 (2008)

    CAS  Article  Google Scholar 

  32. 32

    Sherwood, R. I. et al. Prospective isolation and global gene expression analysis of definitive and visceral endoderm. Dev. Biol. 304, 541–555 (2007)

    CAS  Article  Google Scholar 

  33. 33

    Aiba, K. et al. Defining developmental potency and cell lineage trajectories by expression profiling of differentiating mouse embryonic stem cells. DNA Res. 16, 73–80 (2009)

    CAS  Article  Google Scholar 

  34. 34

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005)

    ADS  CAS  Article  Google Scholar 

  35. 35

    Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genet. 24, 372–376 (2000)

    CAS  Article  Google Scholar 

  36. 36

    Pasini, D., Bracken, A. P., Hansen, J. B., Capillo, M. & Helin, K. The polycomb group protein Suz12 is required for embryonic stem cell differentiation. Mol. Cell. Biol. 27, 3769–3779 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Jiang, H. et al. Role for Dpy-30 in ES cell-fate specification by regulation of H3K4 methylation within bivalent domains. Cell 144, 513–525 (2011)

    CAS  Article  Google Scholar 

  38. 38

    Marson, A. et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 134, 521–533 (2008)

    CAS  Article  Google Scholar 

  39. 39

    Kunarso, G. et al. Transposable elements have rewired the core regulatory network of human embryonic stem cells. Nature Genet. 42, 631–634 (2010)

    CAS  Article  Google Scholar 

  40. 40

    Jiang, J. et al. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nature Cell Biol. 10, 353–360 (2008)

    Article  Google Scholar 

  41. 41

    Geiss, G. K. et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotechnol. 26, 317–325 (2008)

    CAS  Article  Google Scholar 

  42. 42

    Dey, B. K. et al. The histone demethylase KDM5b/JARID1b plays a role in cell fate decisions by blocking terminal differentiation. Mol. Cell. Biol. 28, 5312–5327 (2008)

    CAS  Article  Google Scholar 

  43. 43

    Cloos, P. A., Christensen, J., Agger, K. & Helin, K. Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease. Genes Dev. 22, 1115–1140 (2008)

    CAS  Article  Google Scholar 

  44. 44

    Zappulla, D. C. & Cech, T. R. Yeast telomerase RNA: a flexible scaffold for protein subunits. Proc. Natl Acad. Sci. USA 101, 10024–10029 (2004)

    ADS  CAS  Article  Google Scholar 

  45. 45

    Wutz, A., Rasmussen, T. P. & Jaenisch, R. Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nature Genet. 30, 167–174 (2002)

    CAS  Article  Google Scholar 

  46. 46

    Tsai, M. C. et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science 329, 689–693 (2010)

    ADS  CAS  Article  Google Scholar 

  47. 47

    Meissner, A., Eminli, S. & Jaenisch, R. Derivation and manipulation of murine embryonic stem cells. Methods Mol. Biol. 482, 3–19 (2009)

    CAS  Article  Google Scholar 

  48. 48

    Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998)

    CAS  Article  Google Scholar 

  49. 49

    Avilion, A. A. et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126–140 (2003)

    CAS  Article  Google Scholar 

  50. 50

    Niwa, H., Burdon, T., Chambers, I. & Smith, A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 12, 2048–2060 (1998)

    CAS  Article  Google Scholar 

  51. 51

    Nakatake, Y. et al. Klf4 cooperates with Oct3/4 and Sox2 to activate the Lefty1 core promoter in embryonic stem cells. Mol. Cell. Biol. 26, 7772–7782 (2006)

    CAS  Article  Google Scholar 

  52. 52

    Brons, I. G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007)

    ADS  CAS  Article  Google Scholar 

  53. 53

    Torres-Padilla, M. E., Parfitt, D. E., Kouzarides, T. & Zernicka-Goetz, M. Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature 445, 214–218 (2007)

    ADS  CAS  Article  Google Scholar 

  54. 54

    Gaspar-Maia, A. et al. Chd1 regulates open chromatin and pluripotency of embryonic stem cells. Nature 460, 863–868 (2009)

    ADS  CAS  Article  Google Scholar 

  55. 55

    Dejosez, M. et al. Ronin is essential for embryogenesis and the pluripotency of mouse embryonic stem cells. Cell 133, 1162–1174 (2008)

    CAS  Article  Google Scholar 

  56. 56

    Yuan, P. et al. Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells. Genes Dev. 23, 2507–2520 (2009)

    CAS  Article  Google Scholar 

  57. 57

    Pruitt, K. D., Tatusova, T., Klimke, W. & Maglott, D. R. NCBI Reference Sequences: current status, policy and new initiatives. Nucleic Acids Res. 37, D32–D36 (2009)

    CAS  Article  Google Scholar 

  58. 58

    Yang, Y. H. et al. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15 (2002)

    Article  Google Scholar 

  59. 59

    Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol. Cell 32, 491–502 (2008)

    MathSciNet  CAS  Article  Google Scholar 

  60. 60

    Aiba, K. et al. Defining a developmental path to neural fate by global expression profiling of mouse embryonic stem cells and adult neural stem/progenitor cells. Stem Cells 24, 889–895 (2006)

    CAS  Article  Google Scholar 

  61. 61

    Ying, Q. L., Stavridis, M., Griffiths, D., Li, M. & Smith, A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nature Biotechnol. 21, 183–186 (2003)

    CAS  Article  Google Scholar 

  62. 62

    Morrisey, E. E. et al. GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes Dev. 12, 3579–3590 (1998)

    CAS  Article  Google Scholar 

  63. 63

    Bock, C. et al. Reference maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell 144, 439–452 (2011)

    CAS  Article  Google Scholar 

  64. 64

    Barbie, D. A. et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462, 108–112 (2009)

    ADS  CAS  Article  Google Scholar 

  65. 65

    Lamb, J. et al. The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929–1935 (2006)

    ADS  CAS  Article  Google Scholar 

  66. 66

    Langmead, B., Hansen, K. D. & Leek, J. T. Cloud-scale RNA-sequencing differential expression analysis with Myrna. Genome Biol. 11, R83 (2010)

    Article  Google Scholar 

  67. 67

    Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008)

    Article  Google Scholar 

  68. 68

    Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009)

    ADS  CAS  Article  Google Scholar 

  69. 69

    Katz, Y., Wang, E. T., Airoldi, E. M. & Burge, C. B. Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nature Methods 7, 1009–1015 (2010)

    CAS  Article  Google Scholar 

  70. 70

    Licatalosi, D. D. et al. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464–469 (2008)

    ADS  CAS  Article  Google Scholar 

  71. 71

    Ule, J. et al. CLIP identifies Nova-regulated RNA networks in the brain. Science 302, 1212–1215 (2003)

    ADS  CAS  Article  Google Scholar 

  72. 72

    Wang, Z., Tollervey, J., Briese, M., Turner, D. & Ule, J. CLIP: construction of cDNA libraries for high-throughput sequencing from RNAs cross-linked to proteins in vivo . Methods 48, 287–293 (2009)

    Article  Google Scholar 

Download references


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




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.

Corresponding authors

Correspondence to Mitchell Guttman or Eric S. Lander.

Ethics declarations

Competing interests

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

Supplementary information

Supplementary Figures

The file contains Supplementary Figures 1-17 with legends. (PDF 5868 kb)

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. (ZIP 4048 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Guttman, M., Donaghey, J., Carey, B. et al. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477, 295–300 (2011).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing