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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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Mediator confers the ncRNA-as dependent activation of a heterologous reportera, Schematic representation of genomic control insert (top) or ncRNA-a7 insert (bottom) in luciferase reporter vector driven by a TK-promoter.b, Depletion of ncRNA-a7 reduces the luciferase activity in the ncRNA-a7 luciferase reporter cell lines.c, Depletion of the transcription factors or enhancers in the control or ncRNA-a7 reporter cell lines. The red bar signifies reduced transcription using siRNA against MED12, comparable with siRNA against ncRNA-a7.d, Depletion of different Mediator subunits using the ncRNA-a7 reporter cell lines.e, Depletion of the Mediator subunits in ncRNA-a1 or ncRNA-a3 luciferase reporter cell lines. All data shown are mean ±SEM of three independent experiments. **p<0.01, ***p<0.001 by two-tailed Student’s T-test.
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Figure 1: Mediator confers the ncRNA-as dependent activation of a heterologous reportera, Schematic representation of genomic control insert (top) or ncRNA-a7 insert (bottom) in luciferase reporter vector driven by a TK-promoter.b, Depletion of ncRNA-a7 reduces the luciferase activity in the ncRNA-a7 luciferase reporter cell lines.c, Depletion of the transcription factors or enhancers in the control or ncRNA-a7 reporter cell lines. The red bar signifies reduced transcription using siRNA against MED12, comparable with siRNA against ncRNA-a7.d, Depletion of different Mediator subunits using the ncRNA-a7 reporter cell lines.e, Depletion of the Mediator subunits in ncRNA-a1 or ncRNA-a3 luciferase reporter cell lines. All data shown are mean ±SEM of three independent experiments. **p<0.01, ***p<0.001 by two-tailed Student’s T-test.

Mentions: To define the transcriptional complex(es) that orchestrate the responsiveness of activating lncRNAs, we used stable cell lines expressing the heterologous TK promoter driving luciferase expression fused to ncRNA-a7 controlled by its own natural promoter and depleted factors known to be involved in transcriptional activation and enhancer function. We also developed stable lines expressing control reporters, in which a DNA fragment devoid of any transcriptional activity was substituted for the ncRNA-a7 genomic region (Fig. 1a). Importantly, depletion of ncRNA-a7 using two different siRNAs specifically decreased the transcription of the reporter construct containing the ncRNA-a7 (Fig. 1b). Next, we depleted factors known to be involved in transcriptional activation (Supplementary Fig. 1a) and examined the responsiveness of cell lines harboring the constructs expressing ncRNA-a7 or control constructs devoid of lncRNAs. Interestingly, only depletion of Mediator subunit, MED12 displayed a differential effect on the transcription of the reporter construct containing the ncRNA-a7 (Fig. 1c). Depletion of other factors was either ineffective in changing transcriptional output (see Cdk9, Cyc T, NIPBL or WDR5) or reduced the transcriptional levels for both constructs (see GTF2B, p300, SMC1) (Fig. 1c).


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)

Mediator confers the ncRNA-as dependent activation of a heterologous reportera, Schematic representation of genomic control insert (top) or ncRNA-a7 insert (bottom) in luciferase reporter vector driven by a TK-promoter.b, Depletion of ncRNA-a7 reduces the luciferase activity in the ncRNA-a7 luciferase reporter cell lines.c, Depletion of the transcription factors or enhancers in the control or ncRNA-a7 reporter cell lines. The red bar signifies reduced transcription using siRNA against MED12, comparable with siRNA against ncRNA-a7.d, Depletion of different Mediator subunits using the ncRNA-a7 reporter cell lines.e, Depletion of the Mediator subunits in ncRNA-a1 or ncRNA-a3 luciferase reporter cell lines. All data shown are mean ±SEM of three independent experiments. **p<0.01, ***p<0.001 by two-tailed Student’s T-test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4109059&req=5

Figure 1: Mediator confers the ncRNA-as dependent activation of a heterologous reportera, Schematic representation of genomic control insert (top) or ncRNA-a7 insert (bottom) in luciferase reporter vector driven by a TK-promoter.b, Depletion of ncRNA-a7 reduces the luciferase activity in the ncRNA-a7 luciferase reporter cell lines.c, Depletion of the transcription factors or enhancers in the control or ncRNA-a7 reporter cell lines. The red bar signifies reduced transcription using siRNA against MED12, comparable with siRNA against ncRNA-a7.d, Depletion of different Mediator subunits using the ncRNA-a7 reporter cell lines.e, Depletion of the Mediator subunits in ncRNA-a1 or ncRNA-a3 luciferase reporter cell lines. All data shown are mean ±SEM of three independent experiments. **p<0.01, ***p<0.001 by two-tailed Student’s T-test.
Mentions: To define the transcriptional complex(es) that orchestrate the responsiveness of activating lncRNAs, we used stable cell lines expressing the heterologous TK promoter driving luciferase expression fused to ncRNA-a7 controlled by its own natural promoter and depleted factors known to be involved in transcriptional activation and enhancer function. We also developed stable lines expressing control reporters, in which a DNA fragment devoid of any transcriptional activity was substituted for the ncRNA-a7 genomic region (Fig. 1a). Importantly, depletion of ncRNA-a7 using two different siRNAs specifically decreased the transcription of the reporter construct containing the ncRNA-a7 (Fig. 1b). Next, we depleted factors known to be involved in transcriptional activation (Supplementary Fig. 1a) and examined the responsiveness of cell lines harboring the constructs expressing ncRNA-a7 or control constructs devoid of lncRNAs. Interestingly, only depletion of Mediator subunit, MED12 displayed a differential effect on the transcription of the reporter construct containing the ncRNA-a7 (Fig. 1c). Depletion of other factors was either ineffective in changing transcriptional output (see Cdk9, Cyc T, NIPBL or WDR5) or reduced the transcriptional levels for both constructs (see GTF2B, p300, SMC1) (Fig. 1c).

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