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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 complex and ncRNA-as promote chromatin loopingThe schematic diagrams represent the genomic locus between the ncRNA-a7, SNAI1 and AURKA loci (a), the ncRNA-a3 and TAL1 (d). The top arrows show the position of primers, the digestion sites are shown in the middle and the s1-8, A-G or 1-8 fragments are presented below.The looping events between ncRNAs and its targets were detected between ncRNA-a7 and SNAI1 (b), ncRNA-a7 and Aurka (c) and ncRNA-a3 and TAL1(e) using chromosome conformation capture (3C). Depletion of MED1 or MED12 abolished the loop interaction (b,c,e, middle panels). Knockdown of ncRNA-a7 or ncRNA-a3 reduced the chromosomal looping events (b,c,e, lower panels). The interaction frequency between the anchoring points and distal fragments were determined by Real-time PCR and normalized to BAC templates and control anchors. Each error bar represents ±SEM from three independent experiments, p<0.01 by two-tailed Student’s T-test. Representative gel images of the 3C experiments for AURKA are presented in Supplementary Figure 3.
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Figure 4: Mediator complex and ncRNA-as promote chromatin loopingThe schematic diagrams represent the genomic locus between the ncRNA-a7, SNAI1 and AURKA loci (a), the ncRNA-a3 and TAL1 (d). The top arrows show the position of primers, the digestion sites are shown in the middle and the s1-8, A-G or 1-8 fragments are presented below.The looping events between ncRNAs and its targets were detected between ncRNA-a7 and SNAI1 (b), ncRNA-a7 and Aurka (c) and ncRNA-a3 and TAL1(e) using chromosome conformation capture (3C). Depletion of MED1 or MED12 abolished the loop interaction (b,c,e, middle panels). Knockdown of ncRNA-a7 or ncRNA-a3 reduced the chromosomal looping events (b,c,e, lower panels). The interaction frequency between the anchoring points and distal fragments were determined by Real-time PCR and normalized to BAC templates and control anchors. Each error bar represents ±SEM from three independent experiments, p<0.01 by two-tailed Student’s T-test. Representative gel images of the 3C experiments for AURKA are presented in Supplementary Figure 3.

Mentions: We next asked whether we could detect long-range chromatin looping between the ncRNA-a7 locus and its targets, SNAI1 and AURKA and whether ncRNA-a7 and the Mediator complex have a role in such an association. We performed chromosome conformation capture (3C) to assess the association of ncRNA-a7 with AURKA as well as SNAI1. We also performed a similar analysis between ncRNA-a3 and TAL1 gene. We used anchoring points near the 3’-UTR of ncRNA-a7 or ncRNA-a3 to measure the extent of chromatin looping between the ncRNA-a7 or ncRNA-a3 locus and their targets as shown in Figure 4a, 4d and Supplementary Fig. 3b and c. This analysis revealed a strong association between the ncRNA-a7 locus and the region encompassing the promoter region of SNAI1 (Fig. 4b, upper panel). Moreover, ncRNA-a7 also displayed an interaction with regions near the 5’-end of the AURKA gene that extended into the body of the gene (Fig. 4c, upper panel). There was no detectable association when a control anchor was placed in the genomic region between ncRNA-a7 and AURKA (Fig. 4b and c).


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 complex and ncRNA-as promote chromatin loopingThe schematic diagrams represent the genomic locus between the ncRNA-a7, SNAI1 and AURKA loci (a), the ncRNA-a3 and TAL1 (d). The top arrows show the position of primers, the digestion sites are shown in the middle and the s1-8, A-G or 1-8 fragments are presented below.The looping events between ncRNAs and its targets were detected between ncRNA-a7 and SNAI1 (b), ncRNA-a7 and Aurka (c) and ncRNA-a3 and TAL1(e) using chromosome conformation capture (3C). Depletion of MED1 or MED12 abolished the loop interaction (b,c,e, middle panels). Knockdown of ncRNA-a7 or ncRNA-a3 reduced the chromosomal looping events (b,c,e, lower panels). The interaction frequency between the anchoring points and distal fragments were determined by Real-time PCR and normalized to BAC templates and control anchors. Each error bar represents ±SEM from three independent experiments, p<0.01 by two-tailed Student’s T-test. Representative gel images of the 3C experiments for AURKA are presented in Supplementary Figure 3.
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Figure 4: Mediator complex and ncRNA-as promote chromatin loopingThe schematic diagrams represent the genomic locus between the ncRNA-a7, SNAI1 and AURKA loci (a), the ncRNA-a3 and TAL1 (d). The top arrows show the position of primers, the digestion sites are shown in the middle and the s1-8, A-G or 1-8 fragments are presented below.The looping events between ncRNAs and its targets were detected between ncRNA-a7 and SNAI1 (b), ncRNA-a7 and Aurka (c) and ncRNA-a3 and TAL1(e) using chromosome conformation capture (3C). Depletion of MED1 or MED12 abolished the loop interaction (b,c,e, middle panels). Knockdown of ncRNA-a7 or ncRNA-a3 reduced the chromosomal looping events (b,c,e, lower panels). The interaction frequency between the anchoring points and distal fragments were determined by Real-time PCR and normalized to BAC templates and control anchors. Each error bar represents ±SEM from three independent experiments, p<0.01 by two-tailed Student’s T-test. Representative gel images of the 3C experiments for AURKA are presented in Supplementary Figure 3.
Mentions: We next asked whether we could detect long-range chromatin looping between the ncRNA-a7 locus and its targets, SNAI1 and AURKA and whether ncRNA-a7 and the Mediator complex have a role in such an association. We performed chromosome conformation capture (3C) to assess the association of ncRNA-a7 with AURKA as well as SNAI1. We also performed a similar analysis between ncRNA-a3 and TAL1 gene. We used anchoring points near the 3’-UTR of ncRNA-a7 or ncRNA-a3 to measure the extent of chromatin looping between the ncRNA-a7 or ncRNA-a3 locus and their targets as shown in Figure 4a, 4d and Supplementary Fig. 3b and c. This analysis revealed a strong association between the ncRNA-a7 locus and the region encompassing the promoter region of SNAI1 (Fig. 4b, upper panel). Moreover, ncRNA-a7 also displayed an interaction with regions near the 5’-end of the AURKA gene that extended into the body of the gene (Fig. 4c, upper panel). There was no detectable association when a control anchor was placed in the genomic region between ncRNA-a7 and AURKA (Fig. 4b and c).

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