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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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Interaction of Mediator and activating ncRNAs is disrupted by FG syndrome mutations of MED12a-b, ncRNA-as associate with Mediator complex in vitro. RNA immune-precipitation (RIP) was performed using protein A dynabeads coupled with IgG, MED1 or MED12 antibodies. Identical results were obtained in three independent experiments.c, Purification of Mediator protein complex and associated RNAs. The purified FLAG-MED12 affinity elution was subjected to Superose 6 size-exclusion chromatography and the Mediator subunits were detected by Western blot. The Superose 6 fractions (14-38) are shown on top and molecular mass markers (kilodaltons) are at the bottom.The RNAs in each fraction was extracted and was followed by RT-PCR using specific primers.d, UV cross-link followed by RIP using HEK293T whole cell lysates. The associated RNAs were analyzed by RT-qPCR with individual specific RNA primers. The mean ±SEM are from three independent experiments. ***p<0.001 by two-tailed Student’s T-test.e, Protein sequences of MED12 mutants causing FG syndrome (Opitz-Kaveggia syndrome). Part of the protein sequence of MED12 exon 21 (top panel). The mutated sites indicate the G958E mutant (red) and R961W mutant (blue).f, HEK293T cells were transfected with FLAG-MED12 or mutant constructs. After UV cross-linking, total cell extracts were immunoprecipitated and analyzed by Western blotting.g, Following UV-RIP of wild type and mutant MED12 samples shown in (f), RT-qPCR was performed using transcript specific primers from the lncRNAs shown. The data is a representative of three independent experiments. The two-tailed Student’s T-test, indicated a p<0.001 for ncRNA-a1 and ncRNA-a3, while there was no significant difference for HOTAIR and HOTTIP.
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Figure 3: Interaction of Mediator and activating ncRNAs is disrupted by FG syndrome mutations of MED12a-b, ncRNA-as associate with Mediator complex in vitro. RNA immune-precipitation (RIP) was performed using protein A dynabeads coupled with IgG, MED1 or MED12 antibodies. Identical results were obtained in three independent experiments.c, Purification of Mediator protein complex and associated RNAs. The purified FLAG-MED12 affinity elution was subjected to Superose 6 size-exclusion chromatography and the Mediator subunits were detected by Western blot. The Superose 6 fractions (14-38) are shown on top and molecular mass markers (kilodaltons) are at the bottom.The RNAs in each fraction was extracted and was followed by RT-PCR using specific primers.d, UV cross-link followed by RIP using HEK293T whole cell lysates. The associated RNAs were analyzed by RT-qPCR with individual specific RNA primers. The mean ±SEM are from three independent experiments. ***p<0.001 by two-tailed Student’s T-test.e, Protein sequences of MED12 mutants causing FG syndrome (Opitz-Kaveggia syndrome). Part of the protein sequence of MED12 exon 21 (top panel). The mutated sites indicate the G958E mutant (red) and R961W mutant (blue).f, HEK293T cells were transfected with FLAG-MED12 or mutant constructs. After UV cross-linking, total cell extracts were immunoprecipitated and analyzed by Western blotting.g, Following UV-RIP of wild type and mutant MED12 samples shown in (f), RT-qPCR was performed using transcript specific primers from the lncRNAs shown. The data is a representative of three independent experiments. The two-tailed Student’s T-test, indicated a p<0.001 for ncRNA-a1 and ncRNA-a3, while there was no significant difference for HOTAIR and HOTTIP.

Mentions: We next asked whether ncRNA-a could associate with the Mediator complex. We isolated Mediator using stable cell lines expressing Flag-epitope tagged MED12 and examined its interaction with in vitro transcribed ncRNA-a7. An eluate from a stable Flag-GFP cell line was used as a control (Fig. 3a). While the control primary let7 transcript (prilet7) or the ncRNA, HOTAIR, (a 429 nucleotide RNA with a similar base composition as ncRNA-a7) did not specifically interact with the Mediator complex, we detected a robust and specific association between ncRNA-a7 and the Mediator complex (Fig. 3a). Importantly, Mediator also associated with ncRNA-a1 and ncRNA-a3, two other ncRNA-as that were shown to activate transcription of their neighboring genes 5 (Fig. 3b).


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)

Interaction of Mediator and activating ncRNAs is disrupted by FG syndrome mutations of MED12a-b, ncRNA-as associate with Mediator complex in vitro. RNA immune-precipitation (RIP) was performed using protein A dynabeads coupled with IgG, MED1 or MED12 antibodies. Identical results were obtained in three independent experiments.c, Purification of Mediator protein complex and associated RNAs. The purified FLAG-MED12 affinity elution was subjected to Superose 6 size-exclusion chromatography and the Mediator subunits were detected by Western blot. The Superose 6 fractions (14-38) are shown on top and molecular mass markers (kilodaltons) are at the bottom.The RNAs in each fraction was extracted and was followed by RT-PCR using specific primers.d, UV cross-link followed by RIP using HEK293T whole cell lysates. The associated RNAs were analyzed by RT-qPCR with individual specific RNA primers. The mean ±SEM are from three independent experiments. ***p<0.001 by two-tailed Student’s T-test.e, Protein sequences of MED12 mutants causing FG syndrome (Opitz-Kaveggia syndrome). Part of the protein sequence of MED12 exon 21 (top panel). The mutated sites indicate the G958E mutant (red) and R961W mutant (blue).f, HEK293T cells were transfected with FLAG-MED12 or mutant constructs. After UV cross-linking, total cell extracts were immunoprecipitated and analyzed by Western blotting.g, Following UV-RIP of wild type and mutant MED12 samples shown in (f), RT-qPCR was performed using transcript specific primers from the lncRNAs shown. The data is a representative of three independent experiments. The two-tailed Student’s T-test, indicated a p<0.001 for ncRNA-a1 and ncRNA-a3, while there was no significant difference for HOTAIR and HOTTIP.
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Figure 3: Interaction of Mediator and activating ncRNAs is disrupted by FG syndrome mutations of MED12a-b, ncRNA-as associate with Mediator complex in vitro. RNA immune-precipitation (RIP) was performed using protein A dynabeads coupled with IgG, MED1 or MED12 antibodies. Identical results were obtained in three independent experiments.c, Purification of Mediator protein complex and associated RNAs. The purified FLAG-MED12 affinity elution was subjected to Superose 6 size-exclusion chromatography and the Mediator subunits were detected by Western blot. The Superose 6 fractions (14-38) are shown on top and molecular mass markers (kilodaltons) are at the bottom.The RNAs in each fraction was extracted and was followed by RT-PCR using specific primers.d, UV cross-link followed by RIP using HEK293T whole cell lysates. The associated RNAs were analyzed by RT-qPCR with individual specific RNA primers. The mean ±SEM are from three independent experiments. ***p<0.001 by two-tailed Student’s T-test.e, Protein sequences of MED12 mutants causing FG syndrome (Opitz-Kaveggia syndrome). Part of the protein sequence of MED12 exon 21 (top panel). The mutated sites indicate the G958E mutant (red) and R961W mutant (blue).f, HEK293T cells were transfected with FLAG-MED12 or mutant constructs. After UV cross-linking, total cell extracts were immunoprecipitated and analyzed by Western blotting.g, Following UV-RIP of wild type and mutant MED12 samples shown in (f), RT-qPCR was performed using transcript specific primers from the lncRNAs shown. The data is a representative of three independent experiments. The two-tailed Student’s T-test, indicated a p<0.001 for ncRNA-a1 and ncRNA-a3, while there was no significant difference for HOTAIR and HOTTIP.
Mentions: We next asked whether ncRNA-a could associate with the Mediator complex. We isolated Mediator using stable cell lines expressing Flag-epitope tagged MED12 and examined its interaction with in vitro transcribed ncRNA-a7. An eluate from a stable Flag-GFP cell line was used as a control (Fig. 3a). While the control primary let7 transcript (prilet7) or the ncRNA, HOTAIR, (a 429 nucleotide RNA with a similar base composition as ncRNA-a7) did not specifically interact with the Mediator complex, we detected a robust and specific association between ncRNA-a7 and the Mediator complex (Fig. 3a). Importantly, Mediator also associated with ncRNA-a1 and ncRNA-a3, two other ncRNA-as that were shown to activate transcription of their neighboring genes 5 (Fig. 3b).

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