Activating RNAs associate with Mediator to enhance chromatin architecture and transcription

Abstract

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms1,2,3,4,5,6,7,8. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions8,9,10,11,12,13. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism5,14,15,16. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease-17,18 displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

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Figure 1: Mediator confers the ncRNA-a-dependent activation of a heterologous reporter.
Figure 2: Functional association of Mediator and activating ncRNAs.
Figure 3: Interaction of Mediator and activating ncRNAs is disrupted by FG syndrome mutations of MED12.
Figure 4: Mediator complex and ncRNA-a promote chromatin looping.

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Acknowledgements

We thank Shiekhattar laboratory members for support and discussions. We thank W. Dang (Blobel laboratory) and I. Tempera for suggestions and technical help on 3C experiments. R.S. was partially supported by a cancer core grant from NIH (P30 CA 010815). D.J.T. was supported by a grant from the NCI (R01 CA127364). G.A.B. was supported by 5R37DK058044.

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R.S., F.L. and U.A.O. conceived and designed the overall project with help from G.A.B., M.B., D.J.T. and M.C. F.L. and U.A.O. performed the RNAi screens; F.L. received advice from G.A.B. to execute the 3C experiments. F.L. and M.C. performed the ChIP experiments for Mediator. F.L. performed all experiments regarding the ncRNA association with Mediator and the kinase assays. F.L., M.C. and R.S. analysed the data and wrote the paper.

Corresponding author

Correspondence to Ramin Shiekhattar.

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The authors declare no competing financial interests.

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Lai, F., Orom, U., Cesaroni, M. et al. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494, 497–501 (2013). https://doi.org/10.1038/nature11884

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