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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

Genome-wide mapping of MEG3 lncRNA binding sites.(a) RT–qPCR analysis showing specific enrichment (presentedas percentage of input) of MEG3 but not MALAT1 RNA in the ChOPpull-down assay with MEG3 antisense probes. The ChOP pull-down withGFP antisense oligo, used as a negative control, did not show any enrichmentof MEG3 and MALAT1 RNAs. (b) Genomic tracks showingChOP-seq (MEG3, GFP and input) and ChIP-seq (H3K4me1) intensities,visualized in log scale. The MEG3 binding site is located upstream ofthe TGFBR1 gene (falls within the intron of the COL15A1 gene)and it overlaps with H3K4me1 peaks in BT-549 cells. (c)ChIP–qPCR showing enrichment of H3K27me3 chromatin marks,presented as percentage of input, over the MEG3 peaks associated withthe TGF-β genes in Ctrlsh and MEG3sh cells(±s.d., n=3). (d) Schematic outline ofthe TGFBR1 gene showing MEG3 peaks and the location of 3Cprimers (P1–P9), as indicated by arrows. EcoRI restriction sitesare shown as blue vertical lines. Each error barrepresents ±s.d. from three experiments. Loopingevents between the upstream MEG3 binding site (corresponding to P2primer) and the TGFBR1 promoter detected by 3C–qPCR inCtrlsh and MEG3sh cells. The P values were calculated usingStudent's t-test (two-tailed, two-sample unequal variance),*P<0.05.
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f4: Genome-wide mapping of MEG3 lncRNA binding sites.(a) RT–qPCR analysis showing specific enrichment (presentedas percentage of input) of MEG3 but not MALAT1 RNA in the ChOPpull-down assay with MEG3 antisense probes. The ChOP pull-down withGFP antisense oligo, used as a negative control, did not show any enrichmentof MEG3 and MALAT1 RNAs. (b) Genomic tracks showingChOP-seq (MEG3, GFP and input) and ChIP-seq (H3K4me1) intensities,visualized in log scale. The MEG3 binding site is located upstream ofthe TGFBR1 gene (falls within the intron of the COL15A1 gene)and it overlaps with H3K4me1 peaks in BT-549 cells. (c)ChIP–qPCR showing enrichment of H3K27me3 chromatin marks,presented as percentage of input, over the MEG3 peaks associated withthe TGF-β genes in Ctrlsh and MEG3sh cells(±s.d., n=3). (d) Schematic outline ofthe TGFBR1 gene showing MEG3 peaks and the location of 3Cprimers (P1–P9), as indicated by arrows. EcoRI restriction sitesare shown as blue vertical lines. Each error barrepresents ±s.d. from three experiments. Loopingevents between the upstream MEG3 binding site (corresponding to P2primer) and the TGFBR1 promoter detected by 3C–qPCR inCtrlsh and MEG3sh cells. The P values were calculated usingStudent's t-test (two-tailed, two-sample unequal variance),*P<0.05.

Mentions: We next wanted to address an important question: how does MEG3 target themultiple TGF-β pathway genes in trans? For this purpose, wewanted to fine-map genome-wide MEG3 binding sites using the ChOP methodwith minor modifications. We have previously used ChOP methodology tocharacterize the Kcnq1ot1 lncRNA binding sites on mouse chromosome 7(refs 10, 35). Thismethod is conceptually equivalent to other methods currently used to fine-mapRNA binding sites16171936. We used 15 biotin-labelledantisense DNA oligonucleotides (oligos) spanning across MEG3 RNA (Supplementary Fig. 6a) to ensurerobust capture of MEG3 RNA-associated genomic loci with streptavidinbeads. The ChOP pull-down using MEG3 antisense oligos detected specificenrichment of the MEG3 RNA, but not abundantly expressed nuclear-enrichedMALAT1 lncRNA, whereas pull-down with a biotin probe against greenfluorescence protein (GFP) RNA (with no known target in the human genome), usedas a negative control, detected neither MEG3 nor MALAT1,highlighting the specificity of the ChOP pull-down assay (Fig.4a). We then subjected the ChOP pull-down chromatin material withMEG3 and control probes to high-throughput DNA sequencing. Byconsidering the MEG3-enriched regions over input and nonspecific GFPprobes, we detected 6,837 MEG3-bound genomic regions associated with5,622 genes (Table 2), as identified using the GREATtool37. We found a significant overlap between thederegulated genes from the microarray experiment following MEG3downregulation and the genes associated with MEG3 peaks (300 genes,P<5e−14, hypergeometricdistribution), indicating a functional role of the MEG3 peaks in theregulation of associated genes (Table 2, Supplementary Fig. 6b, Supplementary Data 8 and Supplementary Data 9). We also observed asignificant overlap between the deregulated genes from RNA sequencing or fromboth microarray and RNA sequencing experiments, and the genes associated withMEG3 peaks (Supplementary Fig.6b,c). When we performed network analysis with the 300 deregulatedgenes associated with the MEG3 peaks, we found that TGF-β wasone of the major affected pathways (Supplementary Fig. 7a,b). The majority of the MEG3-bound peaksassociated with the deregulated genes were located distal to the promoter,including genes involved in the TGF-β pathway (Table2, Fig. 4b and Supplementary Fig. 8a-o), suggesting that theMEG3-bound regions may serve as distal regulatory elements, and thatthe MEG3/EZH2 functional interaction contributes to their regulation. Toverify whether the enrichment of the MEG3-bound regions is due to anartefact of direct interaction between the MEG3 genomic locus and theMEG3 peaks identified, we performed ChOP using sense and antisenseoligos. We validated the enrichment of the MEG3 peaks associated with theTGF-β pathway genes in ChOP pull-down with the antisense oligos but notwith the sense oligos. The enrichment with antisense oligos was lost when thechromatin was pretreated with RNase A (Supplementary Fig. 9a). This further suggests that the pull-down withantisense oligos is mediated by MEG3 RNA rather than being the result oftechnical artefacts. To identify MEG3-bound peaks that overlap withputative enhancers in BT-549 cells, we performed H3K4me1 ChIP-seq and overlappedthe H3K4me1 peaks with MEG3 peaks, and found that 662 MEG3 peaksoverlapped with H3K4me1 peaks (H3K4me1/MEG3 peaks) in BT-549 cells(Table 2). The H3K4me1/MEG3 peaks'associated genes showed a significant overlap with the genes that werederegulated upon downregulation of MEG3 and had at least one associatedMEG3 peak (Table 2 and Supplementary Fig. 6c).


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)

Genome-wide mapping of MEG3 lncRNA binding sites.(a) RT–qPCR analysis showing specific enrichment (presentedas percentage of input) of MEG3 but not MALAT1 RNA in the ChOPpull-down assay with MEG3 antisense probes. The ChOP pull-down withGFP antisense oligo, used as a negative control, did not show any enrichmentof MEG3 and MALAT1 RNAs. (b) Genomic tracks showingChOP-seq (MEG3, GFP and input) and ChIP-seq (H3K4me1) intensities,visualized in log scale. The MEG3 binding site is located upstream ofthe TGFBR1 gene (falls within the intron of the COL15A1 gene)and it overlaps with H3K4me1 peaks in BT-549 cells. (c)ChIP–qPCR showing enrichment of H3K27me3 chromatin marks,presented as percentage of input, over the MEG3 peaks associated withthe TGF-β genes in Ctrlsh and MEG3sh cells(±s.d., n=3). (d) Schematic outline ofthe TGFBR1 gene showing MEG3 peaks and the location of 3Cprimers (P1–P9), as indicated by arrows. EcoRI restriction sitesare shown as blue vertical lines. Each error barrepresents ±s.d. from three experiments. Loopingevents between the upstream MEG3 binding site (corresponding to P2primer) and the TGFBR1 promoter detected by 3C–qPCR inCtrlsh and MEG3sh cells. The P values were calculated usingStudent's t-test (two-tailed, two-sample unequal variance),*P<0.05.
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Related In: Results  -  Collection

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f4: Genome-wide mapping of MEG3 lncRNA binding sites.(a) RT–qPCR analysis showing specific enrichment (presentedas percentage of input) of MEG3 but not MALAT1 RNA in the ChOPpull-down assay with MEG3 antisense probes. The ChOP pull-down withGFP antisense oligo, used as a negative control, did not show any enrichmentof MEG3 and MALAT1 RNAs. (b) Genomic tracks showingChOP-seq (MEG3, GFP and input) and ChIP-seq (H3K4me1) intensities,visualized in log scale. The MEG3 binding site is located upstream ofthe TGFBR1 gene (falls within the intron of the COL15A1 gene)and it overlaps with H3K4me1 peaks in BT-549 cells. (c)ChIP–qPCR showing enrichment of H3K27me3 chromatin marks,presented as percentage of input, over the MEG3 peaks associated withthe TGF-β genes in Ctrlsh and MEG3sh cells(±s.d., n=3). (d) Schematic outline ofthe TGFBR1 gene showing MEG3 peaks and the location of 3Cprimers (P1–P9), as indicated by arrows. EcoRI restriction sitesare shown as blue vertical lines. Each error barrepresents ±s.d. from three experiments. Loopingevents between the upstream MEG3 binding site (corresponding to P2primer) and the TGFBR1 promoter detected by 3C–qPCR inCtrlsh and MEG3sh cells. The P values were calculated usingStudent's t-test (two-tailed, two-sample unequal variance),*P<0.05.
Mentions: We next wanted to address an important question: how does MEG3 target themultiple TGF-β pathway genes in trans? For this purpose, wewanted to fine-map genome-wide MEG3 binding sites using the ChOP methodwith minor modifications. We have previously used ChOP methodology tocharacterize the Kcnq1ot1 lncRNA binding sites on mouse chromosome 7(refs 10, 35). Thismethod is conceptually equivalent to other methods currently used to fine-mapRNA binding sites16171936. We used 15 biotin-labelledantisense DNA oligonucleotides (oligos) spanning across MEG3 RNA (Supplementary Fig. 6a) to ensurerobust capture of MEG3 RNA-associated genomic loci with streptavidinbeads. The ChOP pull-down using MEG3 antisense oligos detected specificenrichment of the MEG3 RNA, but not abundantly expressed nuclear-enrichedMALAT1 lncRNA, whereas pull-down with a biotin probe against greenfluorescence protein (GFP) RNA (with no known target in the human genome), usedas a negative control, detected neither MEG3 nor MALAT1,highlighting the specificity of the ChOP pull-down assay (Fig.4a). We then subjected the ChOP pull-down chromatin material withMEG3 and control probes to high-throughput DNA sequencing. Byconsidering the MEG3-enriched regions over input and nonspecific GFPprobes, we detected 6,837 MEG3-bound genomic regions associated with5,622 genes (Table 2), as identified using the GREATtool37. We found a significant overlap between thederegulated genes from the microarray experiment following MEG3downregulation and the genes associated with MEG3 peaks (300 genes,P<5e−14, hypergeometricdistribution), indicating a functional role of the MEG3 peaks in theregulation of associated genes (Table 2, Supplementary Fig. 6b, Supplementary Data 8 and Supplementary Data 9). We also observed asignificant overlap between the deregulated genes from RNA sequencing or fromboth microarray and RNA sequencing experiments, and the genes associated withMEG3 peaks (Supplementary Fig.6b,c). When we performed network analysis with the 300 deregulatedgenes associated with the MEG3 peaks, we found that TGF-β wasone of the major affected pathways (Supplementary Fig. 7a,b). The majority of the MEG3-bound peaksassociated with the deregulated genes were located distal to the promoter,including genes involved in the TGF-β pathway (Table2, Fig. 4b and Supplementary Fig. 8a-o), suggesting that theMEG3-bound regions may serve as distal regulatory elements, and thatthe MEG3/EZH2 functional interaction contributes to their regulation. Toverify whether the enrichment of the MEG3-bound regions is due to anartefact of direct interaction between the MEG3 genomic locus and theMEG3 peaks identified, we performed ChOP using sense and antisenseoligos. We validated the enrichment of the MEG3 peaks associated with theTGF-β pathway genes in ChOP pull-down with the antisense oligos but notwith the sense oligos. The enrichment with antisense oligos was lost when thechromatin was pretreated with RNase A (Supplementary Fig. 9a). This further suggests that the pull-down withantisense oligos is mediated by MEG3 RNA rather than being the result oftechnical artefacts. To identify MEG3-bound peaks that overlap withputative enhancers in BT-549 cells, we performed H3K4me1 ChIP-seq and overlappedthe H3K4me1 peaks with MEG3 peaks, and found that 662 MEG3 peaksoverlapped with H3K4me1 peaks (H3K4me1/MEG3 peaks) in BT-549 cells(Table 2). The H3K4me1/MEG3 peaks'associated genes showed a significant overlap with the genes that werederegulated upon downregulation of MEG3 and had at least one associatedMEG3 peak (Table 2 and Supplementary Fig. 6c).

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