Letter | Published:

Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood

Nature volume 548, pages 343346 (17 August 2017) | Download Citation

  • An Erratum to this article was published on 20 September 2017

Abstract

Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs)1,2, some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms3,4,5,6,7,8. Although some lncRNA loci encode RNAs that act non-locally (in trans)5, there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes—for example, through functions of the lncRNA promoter, transcription, or transcript itself3,6,7,8. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR–Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

BioProject

Gene Expression Omnibus

References

  1. 1.

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

  2. 2.

    et al. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 25, 1915–1927 (2011)

  3. 3.

    et al. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472, 120–124 (2011)

  4. 4.

    et al. The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, 1237973 (2013)

  5. 5.

    et al. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493, 231–235 (2013)

  6. 6.

    et al. Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development. Nature 539, 433–436 (2016)

  7. 7.

    et al. Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature 539, 452–455 (2016)

  8. 8.

    et al. Unlinking an lncRNA from its associated cis element. Mol. Cell 62, 104–110 (2016)

  9. 9.

    et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015)

  10. 10.

    et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 44, D733–D745 (2016)

  11. 11.

    et al. A probability-based approach for the analysis of large-scale RNAi screens. Nat. Methods 4, 847–849 (2007)

  12. 12.

    et al. A melanocyte lineage program confers resistance to MAP kinase pathway inhibition. Nature 504, 138–142 (2013). 10.1038/nature12688

  13. 13.

    , & MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation. Curr. Biol. 18, 311–321 (2008)

  14. 14.

    , , & Regulatory feedback from nascent RNA to chromatin and transcription. Nat. Rev. Mol. Cell Biol. 18, 331–337 (2017)

  15. 15.

    et al. Systematic mapping of functional enhancer–promoter connections with CRISPR interference. Science 354, 769–773 (2016)

  16. 16.

    . et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, aah7111 (2017)

  17. 17.

    et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016)

  18. 18.

    et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016)

  19. 19.

    et al. Genome-scale CRISPR–Cas9 knockout and transcriptional activation screening. Nat. Protocols 12, 828–863 (2017)

  20. 20.

    , , & Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

  21. 21.

    & RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011)

  22. 22.

    , & TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009)

  23. 23.

    et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014). 10.1016/j.cell.2014.11.021

  24. 24.

    ; ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012)

  25. 25.

    & A Hidden Markov Model approach to variation among sites in rate of evolution. Mol. Biol. Evol. 13, 93–104 (1996)

  26. 26.

    et al. A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. Cancer Discov. 4, 816–827 (2014)

  27. 27.

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

  28. 28.

    et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–607 (2012)

Download references

Acknowledgements

We thank M. Guttman, C. M. Johannessen and M. Ghandi for helpful discussions and insights; A. Sayeed, R. Deasy, A. Rotem and B. Izar for generating the primary patient melanoma cell lines; and R. Belliveau, R. Macrae and the Zhang laboratory for support and advice. J.M.E. is supported by the Fannie and John Hertz Foundation. O.A.A. is supported by a Paul and Daisy Soros Fellowship and National Defense Science and Engineering Fellowship. J.S.G. is supported by a DOE Computational Science Graduate Fellowship. N.E.S. is supported by the NIH through NHGRI (R00-HG008171). J.B.W. is supported by the NIH through NIDDK (F32-DK096822). C.P.F. is supported by the National Defense Science and Engineering Graduate Fellowship. E.S.L. is supported by UM1HG008895 and funds from the Broad Institute. F.Z. is a New York Stem Cell Foundation-Robertson Investigator. F.Z. is supported by the NIH through NIMH (5DP1-MH100706 and 1R01-MH110049), NSF, Howard Hughes Medical Institute, the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations; and James and Patricia Poitras, Robert Metcalfe, and David Cheng.

Author information

Author notes

    • Silvana Konermann
    •  & Neville E. Sanjana

    Present addresses: Salk Institute for Biological Studies, La Jolla, California, USA (S.K.); New York Genome Center, New York, New York, USA (N.E.S.); Department of Biology, New York University, New York, New York, USA (N.E.S.).

Affiliations

  1. Department of Biological Engineering, MIT, Cambridge, Massachusetts 02139, USA

    • Julia Joung
    •  & Feng Zhang
  2. Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • Julia Joung
    • , Jesse M. Engreitz
    • , Silvana Konermann
    • , Omar O. Abudayyeh
    • , Vanessa K. Verdine
    • , Francois Aguet
    • , Jonathan S. Gootenberg
    • , Neville E. Sanjana
    • , Jason B. Wright
    • , Charles P. Fulco
    • , Yuen-Yi Tseng
    • , Jesse S. Boehm
    • , Eric S. Lander
    •  & Feng Zhang
  3. McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts 02139, USA

    • Julia Joung
    • , Silvana Konermann
    • , Omar O. Abudayyeh
    • , Vanessa K. Verdine
    • , Jonathan S. Gootenberg
    • , Neville E. Sanjana
    • , Jason B. Wright
    •  & Feng Zhang
  4. Department of Brain and Cognitive Science, MIT, Cambridge, Massachusetts 02139, USA

    • Julia Joung
    • , Silvana Konermann
    • , Omar O. Abudayyeh
    • , Jonathan S. Gootenberg
    • , Neville E. Sanjana
    • , Jason B. Wright
    •  & Feng Zhang
  5. Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts 02139, USA

    • Omar O. Abudayyeh
  6. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Jonathan S. Gootenberg
    • , Charles P. Fulco
    •  & Eric S. Lander
  7. Department of Surgery, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA

    • Charles H. Yoon
  8. Department of Biology, MIT, Cambridge, Massachusetts 02139, USA

    • Eric S. Lander

Authors

  1. Search for Julia Joung in:

  2. Search for Jesse M. Engreitz in:

  3. Search for Silvana Konermann in:

  4. Search for Omar O. Abudayyeh in:

  5. Search for Vanessa K. Verdine in:

  6. Search for Francois Aguet in:

  7. Search for Jonathan S. Gootenberg in:

  8. Search for Neville E. Sanjana in:

  9. Search for Jason B. Wright in:

  10. Search for Charles P. Fulco in:

  11. Search for Yuen-Yi Tseng in:

  12. Search for Charles H. Yoon in:

  13. Search for Jesse S. Boehm in:

  14. Search for Eric S. Lander in:

  15. Search for Feng Zhang in:

Contributions

J.J., S.K. and F.Z. conceived and designed the study. J.J. and S.K. conducted the screen. J.J., V.K.V. and J.S.G. performed validation experiments. N.E.S. and J.B.W. performed ATAC–seq and ChIP experiments. J.J. analysed data. O.O.A. and F.A. analysed clinical datasets. J.M.E., C.P.F. and E.S.L. helped with lncRNA experimental design and interpretation. Y.-Y.T., C.H.Y. and J.S.B. generated primary patient melanoma cell lines. J.J., J.M.E., E.S.L. and F.Z. wrote the paper with help from all authors.

Competing interests

The authors have filed a patent application related to this work. F.Z. is an advisor for Editas Medicine and Horizon Discovery.

Corresponding authors

Correspondence to Eric S. Lander or Feng Zhang.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary notes 1-11.

  2. 2.

    Supplementary Data

    This file contains data blots for figure 3b, and for extended data figures 7a-c and 9a.

Zip files

  1. 1.

    Supplementary Tables

    This file contains Supplementary Tables 1-10 and a Supplementary Table guide.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature23451

Further reading

Comments

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.