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MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures.

Mondal T, Subhash S, Vaid R, Enroth S, Uday S, Reinius B, Mitra S, Mohammed A, James AR, Hoberg E, Moustakas A, Gyllensten U, Jones SJ, Gustafsson CM, Sims AH, Westerlund F, Gorab E, Kanduri C - Nat Commun (2015)

Bottom Line: MEG3 binding sites have GA-rich sequences, which guide MEG3 to the chromatin through RNA-DNA triplex formation.We have found that RNA-DNA triplex structures are widespread and are present over the MEG3 binding sites associated with the TGF-β pathway genes.Our findings suggest that RNA-DNA triplex formation could be a general characteristic of target gene recognition by the chromatin-interacting lncRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, SE-40530 Gothenburg, Sweden.

ABSTRACT
Long noncoding RNAs (lncRNAs) regulate gene expression by association with chromatin, but how they target chromatin remains poorly understood. We have used chromatin RNA immunoprecipitation-coupled high-throughput sequencing to identify 276 lncRNAs enriched in repressive chromatin from breast cancer cells. Using one of the chromatin-interacting lncRNAs, MEG3, we explore the mechanisms by which lncRNAs target chromatin. Here we show that MEG3 and EZH2 share common target genes, including the TGF-β pathway genes. Genome-wide mapping of MEG3 binding sites reveals that MEG3 modulates the activity of TGF-β genes by binding to distal regulatory elements. MEG3 binding sites have GA-rich sequences, which guide MEG3 to the chromatin through RNA-DNA triplex formation. We have found that RNA-DNA triplex structures are widespread and are present over the MEG3 binding sites associated with the TGF-β pathway genes. Our findings suggest that RNA-DNA triplex formation could be a general characteristic of target gene recognition by the chromatin-interacting lncRNAs.

No MeSH data available.


Related in: MedlinePlus

RNA–DNA triplexes are present in vivo.(a) Confocal microscopic images showing immunostaining withanti-triplex dA.2rU antibody (green) in BT-549 cells. The nucleus is stainedwith DAPI (4,6-diamidino-2-phenylindole; blue). Immunostaining with noantibody and secondary antibody were used as negative controls. Scale bar, 5μm. The graph to the right shows quantification of the triplexsignal in cytoplasm and nuclear compartments obtained from thethree-dimensional confocal images. The graph represents the average ofcytoplasmic and nuclear signals from >50 cells in several microscopicfields. The error bars indicate s.e.m. The P value was calculatedusing Student's t-test**P<0.01. (b) RNA–DNA triplexstructures are sensitive to RNase A but are resistant to RNase H invivo. Top panel: immunofluorescent staining of BT-549 cells withanti-triplex dA.2rU antibody (green) with no treatment (left), pretreatedwith RNase A (centre), or pretreated with RNase H (right) as indicated.Middle panel: cells were counterstained with DAPI (blue). Bottom panel:overlay of the triplex signals with DAPI staining. Scale bar, 5μm. (c) Triplex-ChIP–qPCR showing enrichment(presented as percentage of input) of triplex structures over theMEG3 peaks associated with the TGF-β pathway genes(TGFBR1, TGFB2 and SMAD2) in BT-549 cells(±s.d., n=3). Actin was used as a negativecontrol. Chromatin was pretreated with RNase A or RNase H before ChIP.Immunoglobulin G (IgG) was used as an antibody control. (d)Triplex-ChIP–qPCR showing enrichment (presented as percentage ofinput) of triplex structures over the MEG3 peaks associated with theTGF-β pathway genes (TGFBR1, TGFB2 and SMAD2)in Ctrlsh and MEG3sh BT-549 cells (±s.d.,n=3). IgG was used as an antibody control.
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f6: RNA–DNA triplexes are present in vivo.(a) Confocal microscopic images showing immunostaining withanti-triplex dA.2rU antibody (green) in BT-549 cells. The nucleus is stainedwith DAPI (4,6-diamidino-2-phenylindole; blue). Immunostaining with noantibody and secondary antibody were used as negative controls. Scale bar, 5μm. The graph to the right shows quantification of the triplexsignal in cytoplasm and nuclear compartments obtained from thethree-dimensional confocal images. The graph represents the average ofcytoplasmic and nuclear signals from >50 cells in several microscopicfields. The error bars indicate s.e.m. The P value was calculatedusing Student's t-test**P<0.01. (b) RNA–DNA triplexstructures are sensitive to RNase A but are resistant to RNase H invivo. Top panel: immunofluorescent staining of BT-549 cells withanti-triplex dA.2rU antibody (green) with no treatment (left), pretreatedwith RNase A (centre), or pretreated with RNase H (right) as indicated.Middle panel: cells were counterstained with DAPI (blue). Bottom panel:overlay of the triplex signals with DAPI staining. Scale bar, 5μm. (c) Triplex-ChIP–qPCR showing enrichment(presented as percentage of input) of triplex structures over theMEG3 peaks associated with the TGF-β pathway genes(TGFBR1, TGFB2 and SMAD2) in BT-549 cells(±s.d., n=3). Actin was used as a negativecontrol. Chromatin was pretreated with RNase A or RNase H before ChIP.Immunoglobulin G (IgG) was used as an antibody control. (d)Triplex-ChIP–qPCR showing enrichment (presented as percentage ofinput) of triplex structures over the MEG3 peaks associated with theTGF-β pathway genes (TGFBR1, TGFB2 and SMAD2)in Ctrlsh and MEG3sh BT-549 cells (±s.d.,n=3). IgG was used as an antibody control.

Mentions: Our in vitro triplex formation assay with MEG3 RNA TFO and GA-richMEG3 target sequences suggests that RNA–DNA triplexformation could guide MEG3 lncRNA to its target genes across the genome.This raises an intriguing question as to whether RNA–DNA triplexstructures are present in vivo. In order to identify such triplexstructures in BT-549 cells, we wanted to perform immunostaining withanti-triplex dA.2rU antibody, which can detect triplex structures. Thespecificity of the dA.2rU antibody in detecting triplex structures has beenverified by enzyme-linked immunosorbent assay4546. In addition,anti-triplex dA.2rU antibody has been used in immunostaining to detect thetriplex structures on polytene chromosomes, and also in two-cell earlypre-implantation mouse embryos4547. Since the anti-triplexdA.2rU antibody was raised against the triplex derived from homopolymericnucleic acids (poly(rU).poly(dA).poly(rU) complex)46, we wantedto test the ability of the anti-triplex dA.2rU antibody to recognizenon-homopolymeric triplexes, which would be relevant for detecting triplexstructures in vivo. For this, we performed immunodots with DNA triplexesbuilt from poly-purine/poly-pyrimidine sequences, including controls for theantibody reactivity. As expected, the antibodies recognized homopolymerictriplexes containing a poly(dA) backbone and did not bind to nucleic acids whennon-complementary sequences in solutions impeded formation of triple-strandedcomplexes. Also, the antibodies clearly bound to the triplex DNA made withpoly-purine/poly-pyrimidine sequences (Supplementary Fig. 12a), indicating that antibody reactivity is notrestricted to three-stranded configurations assembled with homopolymeric nucleicacids. To detect triplex structures in vivo, immunostaining was performedon BT-549 cells with anti- triplex dA.2rU antibody, and specific staining wasobserved with anti- triplex dA.2rU antibody (Fig. 6a). Theanti-triplex staining was distributed in both the nucleus and the cytoplasm withmore enrichment of the triplex-specific staining in the nuclear compartment(Fig. 6a). Triplex-specific staining was significantlyreduced in the cells treated with RNase A (Fig. 6b, leftand middle panel), but it was resistant to treatment with RNase H, whichspecifically cleaves RNA–DNA hybrids (Fig. 6b,right panel). The triplex staining pattern in BT-549 cells was similar to thestaining of triplex structures that had been detected previously in human cellsusing a monoclonal anti-triplex antibody, Jel 318 (ref. 48 and Supplementary Fig.12b). Although the triplex structures were more enriched in thenucleus, we detected triplex-specific staining in the cytoplasm, which could bedue to recognition of the triplex structures present in mitochondria4950. To test this, we labelled the mitochondria in BT-549 cellswith MitoTracker followed by immunostaining with anti-triplex antibody. Weindeed observed a co-localization of the mitochondrial staining with triplexsignals from cytoplasm, suggesting that a part of the cytoplasmic triplexsignals are contributed by the triplex structures present in mitochondria (Supplementary Fig. 12c). We nextwanted to determine whether the triplex structures present at the MEG3binding sites are associated with the TGF-β pathway genes in BT-549cells. We performed triplex-ChIP with anti-triplex dA.2rU antibody and observedenrichment of the selected MEG3 peaks associated with the TGFBR1,TGFB2 and SMAD2 genes. To check the specificity of theanti-triplex dA.2rU pull-down, we pretreated the chromatin with either RNAse Hor RNase A. RNase A treatment, but not RNase H, treatment resulted in completeloss of triplex enrichment (Fig. 6c). We also performedtriplex-ChIP in MEG3-downregulated BT-549 cells and found a decrease inthe enrichment of the triplex structures over the MEG3 peaks associatedwith the TGFBR1, TGFB2 and SMAD2 genes, suggesting that theMEG3 lncRNA regulate these genes through triplex formation (Fig. 6d).


MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures.

Mondal T, Subhash S, Vaid R, Enroth S, Uday S, Reinius B, Mitra S, Mohammed A, James AR, Hoberg E, Moustakas A, Gyllensten U, Jones SJ, Gustafsson CM, Sims AH, Westerlund F, Gorab E, Kanduri C - Nat Commun (2015)

RNA–DNA triplexes are present in vivo.(a) Confocal microscopic images showing immunostaining withanti-triplex dA.2rU antibody (green) in BT-549 cells. The nucleus is stainedwith DAPI (4,6-diamidino-2-phenylindole; blue). Immunostaining with noantibody and secondary antibody were used as negative controls. Scale bar, 5μm. The graph to the right shows quantification of the triplexsignal in cytoplasm and nuclear compartments obtained from thethree-dimensional confocal images. The graph represents the average ofcytoplasmic and nuclear signals from >50 cells in several microscopicfields. The error bars indicate s.e.m. The P value was calculatedusing Student's t-test**P<0.01. (b) RNA–DNA triplexstructures are sensitive to RNase A but are resistant to RNase H invivo. Top panel: immunofluorescent staining of BT-549 cells withanti-triplex dA.2rU antibody (green) with no treatment (left), pretreatedwith RNase A (centre), or pretreated with RNase H (right) as indicated.Middle panel: cells were counterstained with DAPI (blue). Bottom panel:overlay of the triplex signals with DAPI staining. Scale bar, 5μm. (c) Triplex-ChIP–qPCR showing enrichment(presented as percentage of input) of triplex structures over theMEG3 peaks associated with the TGF-β pathway genes(TGFBR1, TGFB2 and SMAD2) in BT-549 cells(±s.d., n=3). Actin was used as a negativecontrol. Chromatin was pretreated with RNase A or RNase H before ChIP.Immunoglobulin G (IgG) was used as an antibody control. (d)Triplex-ChIP–qPCR showing enrichment (presented as percentage ofinput) of triplex structures over the MEG3 peaks associated with theTGF-β pathway genes (TGFBR1, TGFB2 and SMAD2)in Ctrlsh and MEG3sh BT-549 cells (±s.d.,n=3). IgG was used as an antibody control.
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f6: RNA–DNA triplexes are present in vivo.(a) Confocal microscopic images showing immunostaining withanti-triplex dA.2rU antibody (green) in BT-549 cells. The nucleus is stainedwith DAPI (4,6-diamidino-2-phenylindole; blue). Immunostaining with noantibody and secondary antibody were used as negative controls. Scale bar, 5μm. The graph to the right shows quantification of the triplexsignal in cytoplasm and nuclear compartments obtained from thethree-dimensional confocal images. The graph represents the average ofcytoplasmic and nuclear signals from >50 cells in several microscopicfields. The error bars indicate s.e.m. The P value was calculatedusing Student's t-test**P<0.01. (b) RNA–DNA triplexstructures are sensitive to RNase A but are resistant to RNase H invivo. Top panel: immunofluorescent staining of BT-549 cells withanti-triplex dA.2rU antibody (green) with no treatment (left), pretreatedwith RNase A (centre), or pretreated with RNase H (right) as indicated.Middle panel: cells were counterstained with DAPI (blue). Bottom panel:overlay of the triplex signals with DAPI staining. Scale bar, 5μm. (c) Triplex-ChIP–qPCR showing enrichment(presented as percentage of input) of triplex structures over theMEG3 peaks associated with the TGF-β pathway genes(TGFBR1, TGFB2 and SMAD2) in BT-549 cells(±s.d., n=3). Actin was used as a negativecontrol. Chromatin was pretreated with RNase A or RNase H before ChIP.Immunoglobulin G (IgG) was used as an antibody control. (d)Triplex-ChIP–qPCR showing enrichment (presented as percentage ofinput) of triplex structures over the MEG3 peaks associated with theTGF-β pathway genes (TGFBR1, TGFB2 and SMAD2)in Ctrlsh and MEG3sh BT-549 cells (±s.d.,n=3). IgG was used as an antibody control.
Mentions: Our in vitro triplex formation assay with MEG3 RNA TFO and GA-richMEG3 target sequences suggests that RNA–DNA triplexformation could guide MEG3 lncRNA to its target genes across the genome.This raises an intriguing question as to whether RNA–DNA triplexstructures are present in vivo. In order to identify such triplexstructures in BT-549 cells, we wanted to perform immunostaining withanti-triplex dA.2rU antibody, which can detect triplex structures. Thespecificity of the dA.2rU antibody in detecting triplex structures has beenverified by enzyme-linked immunosorbent assay4546. In addition,anti-triplex dA.2rU antibody has been used in immunostaining to detect thetriplex structures on polytene chromosomes, and also in two-cell earlypre-implantation mouse embryos4547. Since the anti-triplexdA.2rU antibody was raised against the triplex derived from homopolymericnucleic acids (poly(rU).poly(dA).poly(rU) complex)46, we wantedto test the ability of the anti-triplex dA.2rU antibody to recognizenon-homopolymeric triplexes, which would be relevant for detecting triplexstructures in vivo. For this, we performed immunodots with DNA triplexesbuilt from poly-purine/poly-pyrimidine sequences, including controls for theantibody reactivity. As expected, the antibodies recognized homopolymerictriplexes containing a poly(dA) backbone and did not bind to nucleic acids whennon-complementary sequences in solutions impeded formation of triple-strandedcomplexes. Also, the antibodies clearly bound to the triplex DNA made withpoly-purine/poly-pyrimidine sequences (Supplementary Fig. 12a), indicating that antibody reactivity is notrestricted to three-stranded configurations assembled with homopolymeric nucleicacids. To detect triplex structures in vivo, immunostaining was performedon BT-549 cells with anti- triplex dA.2rU antibody, and specific staining wasobserved with anti- triplex dA.2rU antibody (Fig. 6a). Theanti-triplex staining was distributed in both the nucleus and the cytoplasm withmore enrichment of the triplex-specific staining in the nuclear compartment(Fig. 6a). Triplex-specific staining was significantlyreduced in the cells treated with RNase A (Fig. 6b, leftand middle panel), but it was resistant to treatment with RNase H, whichspecifically cleaves RNA–DNA hybrids (Fig. 6b,right panel). The triplex staining pattern in BT-549 cells was similar to thestaining of triplex structures that had been detected previously in human cellsusing a monoclonal anti-triplex antibody, Jel 318 (ref. 48 and Supplementary Fig.12b). Although the triplex structures were more enriched in thenucleus, we detected triplex-specific staining in the cytoplasm, which could bedue to recognition of the triplex structures present in mitochondria4950. To test this, we labelled the mitochondria in BT-549 cellswith MitoTracker followed by immunostaining with anti-triplex antibody. Weindeed observed a co-localization of the mitochondrial staining with triplexsignals from cytoplasm, suggesting that a part of the cytoplasmic triplexsignals are contributed by the triplex structures present in mitochondria (Supplementary Fig. 12c). We nextwanted to determine whether the triplex structures present at the MEG3binding sites are associated with the TGF-β pathway genes in BT-549cells. We performed triplex-ChIP with anti-triplex dA.2rU antibody and observedenrichment of the selected MEG3 peaks associated with the TGFBR1,TGFB2 and SMAD2 genes. To check the specificity of theanti-triplex dA.2rU pull-down, we pretreated the chromatin with either RNAse Hor RNase A. RNase A treatment, but not RNase H, treatment resulted in completeloss of triplex enrichment (Fig. 6c). We also performedtriplex-ChIP in MEG3-downregulated BT-549 cells and found a decrease inthe enrichment of the triplex structures over the MEG3 peaks associatedwith the TGFBR1, TGFB2 and SMAD2 genes, suggesting that theMEG3 lncRNA regulate these genes through triplex formation (Fig. 6d).

Bottom Line: MEG3 binding sites have GA-rich sequences, which guide MEG3 to the chromatin through RNA-DNA triplex formation.We have found that RNA-DNA triplex structures are widespread and are present over the MEG3 binding sites associated with the TGF-β pathway genes.Our findings suggest that RNA-DNA triplex formation could be a general characteristic of target gene recognition by the chromatin-interacting lncRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, SE-40530 Gothenburg, Sweden.

ABSTRACT
Long noncoding RNAs (lncRNAs) regulate gene expression by association with chromatin, but how they target chromatin remains poorly understood. We have used chromatin RNA immunoprecipitation-coupled high-throughput sequencing to identify 276 lncRNAs enriched in repressive chromatin from breast cancer cells. Using one of the chromatin-interacting lncRNAs, MEG3, we explore the mechanisms by which lncRNAs target chromatin. Here we show that MEG3 and EZH2 share common target genes, including the TGF-β pathway genes. Genome-wide mapping of MEG3 binding sites reveals that MEG3 modulates the activity of TGF-β genes by binding to distal regulatory elements. MEG3 binding sites have GA-rich sequences, which guide MEG3 to the chromatin through RNA-DNA triplex formation. We have found that RNA-DNA triplex structures are widespread and are present over the MEG3 binding sites associated with the TGF-β pathway genes. Our findings suggest that RNA-DNA triplex formation could be a general characteristic of target gene recognition by the chromatin-interacting lncRNAs.

No MeSH data available.


Related in: MedlinePlus