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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

Molecular characterization of MEG3 and PRC2 interaction.(a) RNA-fluorescence in situ hybridization showing thedistribution of the MEG3 signal (green) in the nucleus (blue, stainedwith 4,6-diamidino-2-phenylindole). An RNase A-treated sample was used as anegative control. Scale bar, 1 μm. (b)RT–qPCR data showing the distribution of lncRNAs andprotein-coding RNAs in the nuclear and cytoplasmic fractions(±s.d., n=3). (c) RT–qPCRanalysis of MEG3, KCNQ1OT1 and U1SnRNA in EZH2RIP-purified RNA from BT-549 cells. U1SnRNA served as negativecontrol. The enrichment is plotted as percentage of input (±s.d.,n=3). (d) Physical map of the MEG3containing numbered exons showing two T-to-C transitions. The exons in redare constitutively expressed and blue are alternatively spliced exons. Firstconversion is part of exon 3 showing higher expression, whereas the secondconversion is part of exon 4 showing low expression in the nuclear RNAsequencing. (e) In vitro interaction of MEG3 and PRC2.The schematic indicates the exons of the WT MEG3 clone. Left:RT–qPCR showing enrichment of sense WT MEG3 and MEG3carrying deletions (Δ340-348 or Δ345-348 MEG3) inin vitro RNA binding assays. Reaction with antisense WTMEG3 or without purified PRC2 served as negative controls. Thebinding efficiency of MEG3 deletions were presented relative to WTMEG3 (±s.d., n=3). Right:RT–qPCR showing the quantification of input RNAs. (f) Upperpanel: western blot showing EZH2 levels after pull-down with biotinylatedsense WT MEG3, antisense WT MEG3, and Δ345-348MEG3 RNAs incubated with nuclear extract. This is arepresentative data set from several experiments. Lower panel: agarose gelpicture showing input biotin-RNA. (g) RT–qPCR resultshowing the relative enrichment of WT MEG3, Δ340-348 andΔ345-348 MEG3 RNAs in the EZH2-RIP, performed after BT-549cells were transfected with WT and mutant MEG3 plasmids. Data werenormalized to the input RNAs and plotted as percentage of input(±s.d., n=3). To distinguish the endogenousMEG3 from the ectopically expressed MEG3, we designedRT–qPCRs primers, with one primer mapped to the transcribedportion of the vector and the other to MEG3 RNA. EndogenousMEG3 served as positive control and U1SnRNA as negativecontrol.
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f2: Molecular characterization of MEG3 and PRC2 interaction.(a) RNA-fluorescence in situ hybridization showing thedistribution of the MEG3 signal (green) in the nucleus (blue, stainedwith 4,6-diamidino-2-phenylindole). An RNase A-treated sample was used as anegative control. Scale bar, 1 μm. (b)RT–qPCR data showing the distribution of lncRNAs andprotein-coding RNAs in the nuclear and cytoplasmic fractions(±s.d., n=3). (c) RT–qPCRanalysis of MEG3, KCNQ1OT1 and U1SnRNA in EZH2RIP-purified RNA from BT-549 cells. U1SnRNA served as negativecontrol. The enrichment is plotted as percentage of input (±s.d.,n=3). (d) Physical map of the MEG3containing numbered exons showing two T-to-C transitions. The exons in redare constitutively expressed and blue are alternatively spliced exons. Firstconversion is part of exon 3 showing higher expression, whereas the secondconversion is part of exon 4 showing low expression in the nuclear RNAsequencing. (e) In vitro interaction of MEG3 and PRC2.The schematic indicates the exons of the WT MEG3 clone. Left:RT–qPCR showing enrichment of sense WT MEG3 and MEG3carrying deletions (Δ340-348 or Δ345-348 MEG3) inin vitro RNA binding assays. Reaction with antisense WTMEG3 or without purified PRC2 served as negative controls. Thebinding efficiency of MEG3 deletions were presented relative to WTMEG3 (±s.d., n=3). Right:RT–qPCR showing the quantification of input RNAs. (f) Upperpanel: western blot showing EZH2 levels after pull-down with biotinylatedsense WT MEG3, antisense WT MEG3, and Δ345-348MEG3 RNAs incubated with nuclear extract. This is arepresentative data set from several experiments. Lower panel: agarose gelpicture showing input biotin-RNA. (g) RT–qPCR resultshowing the relative enrichment of WT MEG3, Δ340-348 andΔ345-348 MEG3 RNAs in the EZH2-RIP, performed after BT-549cells were transfected with WT and mutant MEG3 plasmids. Data werenormalized to the input RNAs and plotted as percentage of input(±s.d., n=3). To distinguish the endogenousMEG3 from the ectopically expressed MEG3, we designedRT–qPCRs primers, with one primer mapped to the transcribedportion of the vector and the other to MEG3 RNA. EndogenousMEG3 served as positive control and U1SnRNA as negativecontrol.

Mentions: Since MEG3 lncRNA was identified as a repressive chromatin-associated RNAin the ChRIP analysis (Fig. 1d,e), and also as one of thechromatin-interacting RNAs in our previous study involving sucrose-fractionatedchromatin from normal human fibroblasts (HF cells)28, we wereinterested in exploring plausible mechanisms by which MEG3 lncRNArecognizes its target genes. Human MEG3 is an lncRNA of ∼1,700nucleotides with different isoforms generated by alternative splicing. Exons1–3 and 8–9 are common to all isoforms, whereas exons4–7 are present in different combinations31. Insitu RNA hybridization and nuclear–cytoplasmic RNAfractionation experiments indicated that MEG3 is located in the nuclearcompartment (Fig. 2a,b). We checked for the interaction ofMEG3 lncRNA with PRC2 by RIP and found robust enrichment ofMEG3 in the PRC2-interacting RNA fraction (Fig.2c), and its fold enrichment was more or less similar to theenrichment of KCNQ1OT1 lncRNA (Fig. 2c), which wasused as positive control for the RIP experiment.


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)

Molecular characterization of MEG3 and PRC2 interaction.(a) RNA-fluorescence in situ hybridization showing thedistribution of the MEG3 signal (green) in the nucleus (blue, stainedwith 4,6-diamidino-2-phenylindole). An RNase A-treated sample was used as anegative control. Scale bar, 1 μm. (b)RT–qPCR data showing the distribution of lncRNAs andprotein-coding RNAs in the nuclear and cytoplasmic fractions(±s.d., n=3). (c) RT–qPCRanalysis of MEG3, KCNQ1OT1 and U1SnRNA in EZH2RIP-purified RNA from BT-549 cells. U1SnRNA served as negativecontrol. The enrichment is plotted as percentage of input (±s.d.,n=3). (d) Physical map of the MEG3containing numbered exons showing two T-to-C transitions. The exons in redare constitutively expressed and blue are alternatively spliced exons. Firstconversion is part of exon 3 showing higher expression, whereas the secondconversion is part of exon 4 showing low expression in the nuclear RNAsequencing. (e) In vitro interaction of MEG3 and PRC2.The schematic indicates the exons of the WT MEG3 clone. Left:RT–qPCR showing enrichment of sense WT MEG3 and MEG3carrying deletions (Δ340-348 or Δ345-348 MEG3) inin vitro RNA binding assays. Reaction with antisense WTMEG3 or without purified PRC2 served as negative controls. Thebinding efficiency of MEG3 deletions were presented relative to WTMEG3 (±s.d., n=3). Right:RT–qPCR showing the quantification of input RNAs. (f) Upperpanel: western blot showing EZH2 levels after pull-down with biotinylatedsense WT MEG3, antisense WT MEG3, and Δ345-348MEG3 RNAs incubated with nuclear extract. This is arepresentative data set from several experiments. Lower panel: agarose gelpicture showing input biotin-RNA. (g) RT–qPCR resultshowing the relative enrichment of WT MEG3, Δ340-348 andΔ345-348 MEG3 RNAs in the EZH2-RIP, performed after BT-549cells were transfected with WT and mutant MEG3 plasmids. Data werenormalized to the input RNAs and plotted as percentage of input(±s.d., n=3). To distinguish the endogenousMEG3 from the ectopically expressed MEG3, we designedRT–qPCRs primers, with one primer mapped to the transcribedportion of the vector and the other to MEG3 RNA. EndogenousMEG3 served as positive control and U1SnRNA as negativecontrol.
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Related In: Results  -  Collection

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f2: Molecular characterization of MEG3 and PRC2 interaction.(a) RNA-fluorescence in situ hybridization showing thedistribution of the MEG3 signal (green) in the nucleus (blue, stainedwith 4,6-diamidino-2-phenylindole). An RNase A-treated sample was used as anegative control. Scale bar, 1 μm. (b)RT–qPCR data showing the distribution of lncRNAs andprotein-coding RNAs in the nuclear and cytoplasmic fractions(±s.d., n=3). (c) RT–qPCRanalysis of MEG3, KCNQ1OT1 and U1SnRNA in EZH2RIP-purified RNA from BT-549 cells. U1SnRNA served as negativecontrol. The enrichment is plotted as percentage of input (±s.d.,n=3). (d) Physical map of the MEG3containing numbered exons showing two T-to-C transitions. The exons in redare constitutively expressed and blue are alternatively spliced exons. Firstconversion is part of exon 3 showing higher expression, whereas the secondconversion is part of exon 4 showing low expression in the nuclear RNAsequencing. (e) In vitro interaction of MEG3 and PRC2.The schematic indicates the exons of the WT MEG3 clone. Left:RT–qPCR showing enrichment of sense WT MEG3 and MEG3carrying deletions (Δ340-348 or Δ345-348 MEG3) inin vitro RNA binding assays. Reaction with antisense WTMEG3 or without purified PRC2 served as negative controls. Thebinding efficiency of MEG3 deletions were presented relative to WTMEG3 (±s.d., n=3). Right:RT–qPCR showing the quantification of input RNAs. (f) Upperpanel: western blot showing EZH2 levels after pull-down with biotinylatedsense WT MEG3, antisense WT MEG3, and Δ345-348MEG3 RNAs incubated with nuclear extract. This is arepresentative data set from several experiments. Lower panel: agarose gelpicture showing input biotin-RNA. (g) RT–qPCR resultshowing the relative enrichment of WT MEG3, Δ340-348 andΔ345-348 MEG3 RNAs in the EZH2-RIP, performed after BT-549cells were transfected with WT and mutant MEG3 plasmids. Data werenormalized to the input RNAs and plotted as percentage of input(±s.d., n=3). To distinguish the endogenousMEG3 from the ectopically expressed MEG3, we designedRT–qPCRs primers, with one primer mapped to the transcribedportion of the vector and the other to MEG3 RNA. EndogenousMEG3 served as positive control and U1SnRNA as negativecontrol.
Mentions: Since MEG3 lncRNA was identified as a repressive chromatin-associated RNAin the ChRIP analysis (Fig. 1d,e), and also as one of thechromatin-interacting RNAs in our previous study involving sucrose-fractionatedchromatin from normal human fibroblasts (HF cells)28, we wereinterested in exploring plausible mechanisms by which MEG3 lncRNArecognizes its target genes. Human MEG3 is an lncRNA of ∼1,700nucleotides with different isoforms generated by alternative splicing. Exons1–3 and 8–9 are common to all isoforms, whereas exons4–7 are present in different combinations31. Insitu RNA hybridization and nuclear–cytoplasmic RNAfractionation experiments indicated that MEG3 is located in the nuclearcompartment (Fig. 2a,b). We checked for the interaction ofMEG3 lncRNA with PRC2 by RIP and found robust enrichment ofMEG3 in the PRC2-interacting RNA fraction (Fig.2c), and its fold enrichment was more or less similar to theenrichment of KCNQ1OT1 lncRNA (Fig. 2c), which wasused as positive control for the RIP experiment.

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