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Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

Lai F, Orom UA, Cesaroni M, Beringer M, Taatjes DJ, Blobel GA, Shiekhattar R - Nature (2013)

Bottom Line: We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10.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.

View Article: PubMed Central - PubMed

Affiliation: The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. 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 functions. 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 mechanism. 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- 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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Functional association of Mediator and activating ncRNAsa, Knockdowns of ncRNA-a7 or Mediator subunits decrease SNAI1, AURKA expression. Depletion of ncRNA-a7 diminished SNAI1 and AURKA expression by Real-time PCR (top red bars). Similarly knockdowns of MED1 (green bars) or MED12 (yellow bars) reduce SNAI1 or AURKA expression.b, Knockdowns of ncRNA-a3 or Mediator subunits decrease TAL1 expression. Expression of TAL1 or control STIL following knockdowns of ncRNA-a3 (red bars), MED1 (green bars) and MED12 (yellow bars) are shown.c-d, Knockdowns of ncRNA-a7 (c) or ncRNA-a3 (d) reduce the genomic occupancy of MED1, MED12 (top panels) or RNAPII (bottom panels) on SNAI1, AURKA (c) or TAL1 (d) in A549 cells.e-f, Activating ncRNAs specifically stimulate Mediator kinase activity toward histone H3.1 substrate in vitro. Quantification of kinase assay following addition of ncRNA-a7 (e) or ncRNA-a3 (f). Error bars represent ±SEM (n=3), p<0.01 by two-tailed Student’s T-test.The mean ±SEM for all results represent three independent experiments. *p<0.05, **p<0.01, ***p<0.001 by two-tailed Student’s T-test.
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Figure 2: Functional association of Mediator and activating ncRNAsa, Knockdowns of ncRNA-a7 or Mediator subunits decrease SNAI1, AURKA expression. Depletion of ncRNA-a7 diminished SNAI1 and AURKA expression by Real-time PCR (top red bars). Similarly knockdowns of MED1 (green bars) or MED12 (yellow bars) reduce SNAI1 or AURKA expression.b, Knockdowns of ncRNA-a3 or Mediator subunits decrease TAL1 expression. Expression of TAL1 or control STIL following knockdowns of ncRNA-a3 (red bars), MED1 (green bars) and MED12 (yellow bars) are shown.c-d, Knockdowns of ncRNA-a7 (c) or ncRNA-a3 (d) reduce the genomic occupancy of MED1, MED12 (top panels) or RNAPII (bottom panels) on SNAI1, AURKA (c) or TAL1 (d) in A549 cells.e-f, Activating ncRNAs specifically stimulate Mediator kinase activity toward histone H3.1 substrate in vitro. Quantification of kinase assay following addition of ncRNA-a7 (e) or ncRNA-a3 (f). Error bars represent ±SEM (n=3), p<0.01 by two-tailed Student’s T-test.The mean ±SEM for all results represent three independent experiments. *p<0.05, **p<0.01, ***p<0.001 by two-tailed Student’s T-test.

Mentions: We next depleted the Mediator subunits and assessed their effect on ncRNA-as targets in vivo. We had previously found that ncRNA-a7 and ncRNA-a3 regulate SNAI1 and TAL1 genes, respectively 5. Moreover, using gene expression arrays, we had observed a profound decrease in AURKA transcription when ncRNA-a7 was depleted 5. We confirmed these results by depleting ncRNA-a7 and ncRNA-a3 which led to a decrease in SNAI1, AURKA and TAL1 transcript levels using real-time PCR (Fig. 2a and b). We next depleted two different subunits of the Mediator complex MED1 and MED12 and assessed the SNAI1, AURKA, and TAL1 transcript levels (Supplementary Fig. 1b). Importantly, depletion of Mediator subunits decreased transcription of all three targets similar to that shown with depletion of ncRNA as (Fig. 2a and b, the affected genes are depicted by a black bar). Interestingly depletion of Mediator subunits did not affect the transcript levels of genes that are not regulated by ncRNA-a (UBE2V1, CSTF, STIL, depicted by a white bar in Fig. 2a and b). However, while ncRNA-a7 transcription was not affected following Mediator depletion, there was a decrease in ncRNA-a3 levels (Fig. 2a and b).


Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

Lai F, Orom UA, Cesaroni M, Beringer M, Taatjes DJ, Blobel GA, Shiekhattar R - Nature (2013)

Functional association of Mediator and activating ncRNAsa, Knockdowns of ncRNA-a7 or Mediator subunits decrease SNAI1, AURKA expression. Depletion of ncRNA-a7 diminished SNAI1 and AURKA expression by Real-time PCR (top red bars). Similarly knockdowns of MED1 (green bars) or MED12 (yellow bars) reduce SNAI1 or AURKA expression.b, Knockdowns of ncRNA-a3 or Mediator subunits decrease TAL1 expression. Expression of TAL1 or control STIL following knockdowns of ncRNA-a3 (red bars), MED1 (green bars) and MED12 (yellow bars) are shown.c-d, Knockdowns of ncRNA-a7 (c) or ncRNA-a3 (d) reduce the genomic occupancy of MED1, MED12 (top panels) or RNAPII (bottom panels) on SNAI1, AURKA (c) or TAL1 (d) in A549 cells.e-f, Activating ncRNAs specifically stimulate Mediator kinase activity toward histone H3.1 substrate in vitro. Quantification of kinase assay following addition of ncRNA-a7 (e) or ncRNA-a3 (f). Error bars represent ±SEM (n=3), p<0.01 by two-tailed Student’s T-test.The mean ±SEM for all results represent three independent experiments. *p<0.05, **p<0.01, ***p<0.001 by two-tailed Student’s T-test.
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Figure 2: Functional association of Mediator and activating ncRNAsa, Knockdowns of ncRNA-a7 or Mediator subunits decrease SNAI1, AURKA expression. Depletion of ncRNA-a7 diminished SNAI1 and AURKA expression by Real-time PCR (top red bars). Similarly knockdowns of MED1 (green bars) or MED12 (yellow bars) reduce SNAI1 or AURKA expression.b, Knockdowns of ncRNA-a3 or Mediator subunits decrease TAL1 expression. Expression of TAL1 or control STIL following knockdowns of ncRNA-a3 (red bars), MED1 (green bars) and MED12 (yellow bars) are shown.c-d, Knockdowns of ncRNA-a7 (c) or ncRNA-a3 (d) reduce the genomic occupancy of MED1, MED12 (top panels) or RNAPII (bottom panels) on SNAI1, AURKA (c) or TAL1 (d) in A549 cells.e-f, Activating ncRNAs specifically stimulate Mediator kinase activity toward histone H3.1 substrate in vitro. Quantification of kinase assay following addition of ncRNA-a7 (e) or ncRNA-a3 (f). Error bars represent ±SEM (n=3), p<0.01 by two-tailed Student’s T-test.The mean ±SEM for all results represent three independent experiments. *p<0.05, **p<0.01, ***p<0.001 by two-tailed Student’s T-test.
Mentions: We next depleted the Mediator subunits and assessed their effect on ncRNA-as targets in vivo. We had previously found that ncRNA-a7 and ncRNA-a3 regulate SNAI1 and TAL1 genes, respectively 5. Moreover, using gene expression arrays, we had observed a profound decrease in AURKA transcription when ncRNA-a7 was depleted 5. We confirmed these results by depleting ncRNA-a7 and ncRNA-a3 which led to a decrease in SNAI1, AURKA and TAL1 transcript levels using real-time PCR (Fig. 2a and b). We next depleted two different subunits of the Mediator complex MED1 and MED12 and assessed the SNAI1, AURKA, and TAL1 transcript levels (Supplementary Fig. 1b). Importantly, depletion of Mediator subunits decreased transcription of all three targets similar to that shown with depletion of ncRNA as (Fig. 2a and b, the affected genes are depicted by a black bar). Interestingly depletion of Mediator subunits did not affect the transcript levels of genes that are not regulated by ncRNA-a (UBE2V1, CSTF, STIL, depicted by a white bar in Fig. 2a and b). However, while ncRNA-a7 transcription was not affected following Mediator depletion, there was a decrease in ncRNA-a3 levels (Fig. 2a and b).

Bottom Line: We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10.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.

View Article: PubMed Central - PubMed

Affiliation: The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. 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 functions. 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 mechanism. 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- 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.

Show MeSH
Related in: MedlinePlus