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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

MEG3 lncRNA regulates its target genes through triplex structureformation.(a) Predicted GA-rich motifs enriched in all MEG3 peaks andpeaks associated with deregulated genes using MEME-ChIP. (b) Numberof TrTS over the MEG3 peak summits and neighbouring regions,predicted by Triplexator41. (c) The schematic showsthe MEG3 TFO used in the triplex assays. The exons are colour-codedas described before. (d) Electrophoretic mobility shift assay.End-labelled dsDNA oligos (sequences provided in the schematic with genename) were incubated alone (lane 1) or with increasing concentrations ofMEG3 ssRNA TFO (lanes 2 and 3: shift indicated with arrow) orwith increasing concentrations of control ssRNA oligo (lanes 8 and 9). dsDNAoligos were incubated with MEG3 TFO and treated with either RNase A(lane 4) or RNase H (lane 5). dsDNA oligos were incubated with MEG3ssRNA TFO in the presence of either unlabelled specific competitor (lane 6)or nonspecific competitor (lane 7). (e) TGFBR1-associatedMEG3 peak sequence and its mutated version (the changednucleotides are in red) were incubated alone (lanes 1 and 7) or withMEG3 TFO. Arrow indicates complex formation. (f,g)Enrichment of MEG3 peak sequences using biotin- and psoralen-labelledMEG3 TFO. RNase H-treated lysates were used to capture thelabelled MEG3 TFO using streptavidin beads. The enrichment ofMEG3 peaks is presented as the ratio between MEG3 TFO andcontrol oligo (±s.d., n=3). (h)RT–qPCR analysis of gene expression in BT-549 cells transfectedwith either MEG3 TFO or control RNA oligo. Expression in MEG3TFO presented relative to the control oligo (±s.d.,n=3). *P<0.05, Student'st-test (two-tailed, two-sample unequal variance). (i) Leftpanel: CD spectra of a 1:1 mixture of TGFB2 dsDNA and MEG3 TFO(ssRNA) are shown in black, and TGFB2 dsDNA and the control ssRNA areshown in red. Right panel: the sum of the individual CD spectra forTGFB2 dsDNA and MEG3 TFO (ssRNA) is shown in black, andthe sum of the individual CD spectra for TGFB2 dsDNA and the controlssRNA is shown in red. Inset in the left and right panel shows thedifference between the two spectra.
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f5: MEG3 lncRNA regulates its target genes through triplex structureformation.(a) Predicted GA-rich motifs enriched in all MEG3 peaks andpeaks associated with deregulated genes using MEME-ChIP. (b) Numberof TrTS over the MEG3 peak summits and neighbouring regions,predicted by Triplexator41. (c) The schematic showsthe MEG3 TFO used in the triplex assays. The exons are colour-codedas described before. (d) Electrophoretic mobility shift assay.End-labelled dsDNA oligos (sequences provided in the schematic with genename) were incubated alone (lane 1) or with increasing concentrations ofMEG3 ssRNA TFO (lanes 2 and 3: shift indicated with arrow) orwith increasing concentrations of control ssRNA oligo (lanes 8 and 9). dsDNAoligos were incubated with MEG3 TFO and treated with either RNase A(lane 4) or RNase H (lane 5). dsDNA oligos were incubated with MEG3ssRNA TFO in the presence of either unlabelled specific competitor (lane 6)or nonspecific competitor (lane 7). (e) TGFBR1-associatedMEG3 peak sequence and its mutated version (the changednucleotides are in red) were incubated alone (lanes 1 and 7) or withMEG3 TFO. Arrow indicates complex formation. (f,g)Enrichment of MEG3 peak sequences using biotin- and psoralen-labelledMEG3 TFO. RNase H-treated lysates were used to capture thelabelled MEG3 TFO using streptavidin beads. The enrichment ofMEG3 peaks is presented as the ratio between MEG3 TFO andcontrol oligo (±s.d., n=3). (h)RT–qPCR analysis of gene expression in BT-549 cells transfectedwith either MEG3 TFO or control RNA oligo. Expression in MEG3TFO presented relative to the control oligo (±s.d.,n=3). *P<0.05, Student'st-test (two-tailed, two-sample unequal variance). (i) Leftpanel: CD spectra of a 1:1 mixture of TGFB2 dsDNA and MEG3 TFO(ssRNA) are shown in black, and TGFB2 dsDNA and the control ssRNA areshown in red. Right panel: the sum of the individual CD spectra forTGFB2 dsDNA and MEG3 TFO (ssRNA) is shown in black, andthe sum of the individual CD spectra for TGFB2 dsDNA and the controlssRNA is shown in red. Inset in the left and right panel shows thedifference between the two spectra.

Mentions: We next tried to investigate the mechanisms that facilitate how MEG3lncRNA selects its target regions across the genome. First, we looked for commonsequence motifs enriched in the MEG3-bound genomic regions and identifieda strong GA-rich sequence motif that was overrepresented among the 6,837MEG3 peak summits (motif e-value:1.7e−976) (Fig. 5a). TheGA-rich motif was also overrepresented among the 532 MEG3 peaks (motife-value: 6.3e−904) associated with theMEG3-deregulated genes (Fig. 5a), suggestingthat the GA-rich repeat may play a functional role in targeting of theMEG3 RNA to chromatin. Interestingly, by using the ChIRP technique,similar GA-enriched motifs were identified among the binding sites of thechromatin-modulating RNAs roX2 and HOTAIR, indicating thatGA-enriched motifs may play an important role in the targeting of lncRNAs acrossthe genome19. Previously, several studies using differenttechniques have shown that GA-rich homopurine sequences can form triplexstructures383940. Overrepresentation of GA-rich sequencesamong the genomic binding sites of the lncRNAs analysed (MEG3,HOTAIR and roX) raises the possibility that the lncRNAs may berecruited to their target genes via RNA–DNA triplex formation. Byusing Triplexator software41 (which can predict triplex targetsites, TrTS), we found a greater number of the predicted TrTS in the MEG3peak summit (±200 bp from the centre of the peak) than theflanking sequences (200 bp upstream and 200 bp downstreamof the peak summit; Fig. 5b). Triplexator was also used toscan for triplex-forming oligonucleotides (TFOs) within the MEG3 RNA, andseveral TFOs with high scores were detected. Interestingly, the TFOs with highscores are also enriched with GA-rich sequences (Table3), indicating that the GA-rich sequences from target genes andMEG3 RNA could form triplex structures by forming Hoogsteen bondsbetween RNA and DNA. To test the ability of MEG3 lncRNA to form triplexstructures, we used a 20-nucleotide-long GA-rich RNA oligo (hereon referred toas MEG3 TFO) located at the 5′-end of the MEG3 RNA andits sequence overlap with the TFOs (TFO1, TFO2 and TFO3) with high score thatwere identified by Triplexator (Fig. 5c and Table 3). Using electrophoretic mobility shift assay, wetested the triplex-forming ability of the MEG3 TFO (single-stranded RNA,ssRNA) with the GA-rich (double-stranded DNA, dsDNA) MEG3 peak sequencesassociated with the selected TGF-β pathway target genes (TGFBR1,TGFB2 and SMAD2) in vitro (Fig.5d). Consistent with the Triplexator predictions, we observed a shiftin the end-labelled GA-rich dsDNA sequences when incubated with increasingconcentrations of the MEG3 TFO, indicating triplex formation between theMEG3 TFO and the GA-rich MEG3 peak summits (Fig. 5d, compare lane 1 with lanes 2 and 3), but not with a controlRNA oligo selected from the MEG3 lncRNA with no GA bias (Fig. 5d, compare lane 1 with lanes 8 and 9). The triplex structureswere sensitive to RNase A treatment but were resistant to RNase H digestion(Fig. 5d, lanes 4 and 5, respectively), while an invitro formed RNA–DNA hybrid was digested by RNase H (Supplementary Fig. 10a). Theseresults together suggest that the observed shift was not because ofWatson–Crick RNA–DNA pairing. We also observed that theseshifts were affected when specific competitor (the same GA-rich dsDNA oligo,unlabelled) was used but were unaffected by nonspecific competitor (unlabelledcontrol dsDNA oligo; Fig. 5d, compare lane 6 with 7). Wedid not observe any complex formation between MEG3 TFO incubated withend-labelled control DNA sequences (with no GA bias) or control RNA incubatedwith control DNA sequences (SupplementaryFig. 10b,c). To further check the specificity of the interactionbetween MEG3 TFO and GA-rich DNA sequences, we mutated the core sequencesof the TGFBR1-associated MEG3 peak and found that triplexformation was compromised between the mutant TGFBR1 dsDNA oligo and theMEG3 TFO (Fig. 5e). These observations suggestthat MEG3 may be recruited to genomic loci through the formation ofRNA–DNA triplex structures. We predicted the triplex-forming abilityof GA-rich motifs of another chromatin-interacting lncRNA, HOTAIR, andfound more Triplexator-predicted TrTS from the HOTAIR summit regions thanfrom the neighbouring sequences (Supplementary Fig. 10d,e).


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)

MEG3 lncRNA regulates its target genes through triplex structureformation.(a) Predicted GA-rich motifs enriched in all MEG3 peaks andpeaks associated with deregulated genes using MEME-ChIP. (b) Numberof TrTS over the MEG3 peak summits and neighbouring regions,predicted by Triplexator41. (c) The schematic showsthe MEG3 TFO used in the triplex assays. The exons are colour-codedas described before. (d) Electrophoretic mobility shift assay.End-labelled dsDNA oligos (sequences provided in the schematic with genename) were incubated alone (lane 1) or with increasing concentrations ofMEG3 ssRNA TFO (lanes 2 and 3: shift indicated with arrow) orwith increasing concentrations of control ssRNA oligo (lanes 8 and 9). dsDNAoligos were incubated with MEG3 TFO and treated with either RNase A(lane 4) or RNase H (lane 5). dsDNA oligos were incubated with MEG3ssRNA TFO in the presence of either unlabelled specific competitor (lane 6)or nonspecific competitor (lane 7). (e) TGFBR1-associatedMEG3 peak sequence and its mutated version (the changednucleotides are in red) were incubated alone (lanes 1 and 7) or withMEG3 TFO. Arrow indicates complex formation. (f,g)Enrichment of MEG3 peak sequences using biotin- and psoralen-labelledMEG3 TFO. RNase H-treated lysates were used to capture thelabelled MEG3 TFO using streptavidin beads. The enrichment ofMEG3 peaks is presented as the ratio between MEG3 TFO andcontrol oligo (±s.d., n=3). (h)RT–qPCR analysis of gene expression in BT-549 cells transfectedwith either MEG3 TFO or control RNA oligo. Expression in MEG3TFO presented relative to the control oligo (±s.d.,n=3). *P<0.05, Student'st-test (two-tailed, two-sample unequal variance). (i) Leftpanel: CD spectra of a 1:1 mixture of TGFB2 dsDNA and MEG3 TFO(ssRNA) are shown in black, and TGFB2 dsDNA and the control ssRNA areshown in red. Right panel: the sum of the individual CD spectra forTGFB2 dsDNA and MEG3 TFO (ssRNA) is shown in black, andthe sum of the individual CD spectra for TGFB2 dsDNA and the controlssRNA is shown in red. Inset in the left and right panel shows thedifference between the two spectra.
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f5: MEG3 lncRNA regulates its target genes through triplex structureformation.(a) Predicted GA-rich motifs enriched in all MEG3 peaks andpeaks associated with deregulated genes using MEME-ChIP. (b) Numberof TrTS over the MEG3 peak summits and neighbouring regions,predicted by Triplexator41. (c) The schematic showsthe MEG3 TFO used in the triplex assays. The exons are colour-codedas described before. (d) Electrophoretic mobility shift assay.End-labelled dsDNA oligos (sequences provided in the schematic with genename) were incubated alone (lane 1) or with increasing concentrations ofMEG3 ssRNA TFO (lanes 2 and 3: shift indicated with arrow) orwith increasing concentrations of control ssRNA oligo (lanes 8 and 9). dsDNAoligos were incubated with MEG3 TFO and treated with either RNase A(lane 4) or RNase H (lane 5). dsDNA oligos were incubated with MEG3ssRNA TFO in the presence of either unlabelled specific competitor (lane 6)or nonspecific competitor (lane 7). (e) TGFBR1-associatedMEG3 peak sequence and its mutated version (the changednucleotides are in red) were incubated alone (lanes 1 and 7) or withMEG3 TFO. Arrow indicates complex formation. (f,g)Enrichment of MEG3 peak sequences using biotin- and psoralen-labelledMEG3 TFO. RNase H-treated lysates were used to capture thelabelled MEG3 TFO using streptavidin beads. The enrichment ofMEG3 peaks is presented as the ratio between MEG3 TFO andcontrol oligo (±s.d., n=3). (h)RT–qPCR analysis of gene expression in BT-549 cells transfectedwith either MEG3 TFO or control RNA oligo. Expression in MEG3TFO presented relative to the control oligo (±s.d.,n=3). *P<0.05, Student'st-test (two-tailed, two-sample unequal variance). (i) Leftpanel: CD spectra of a 1:1 mixture of TGFB2 dsDNA and MEG3 TFO(ssRNA) are shown in black, and TGFB2 dsDNA and the control ssRNA areshown in red. Right panel: the sum of the individual CD spectra forTGFB2 dsDNA and MEG3 TFO (ssRNA) is shown in black, andthe sum of the individual CD spectra for TGFB2 dsDNA and the controlssRNA is shown in red. Inset in the left and right panel shows thedifference between the two spectra.
Mentions: We next tried to investigate the mechanisms that facilitate how MEG3lncRNA selects its target regions across the genome. First, we looked for commonsequence motifs enriched in the MEG3-bound genomic regions and identifieda strong GA-rich sequence motif that was overrepresented among the 6,837MEG3 peak summits (motif e-value:1.7e−976) (Fig. 5a). TheGA-rich motif was also overrepresented among the 532 MEG3 peaks (motife-value: 6.3e−904) associated with theMEG3-deregulated genes (Fig. 5a), suggestingthat the GA-rich repeat may play a functional role in targeting of theMEG3 RNA to chromatin. Interestingly, by using the ChIRP technique,similar GA-enriched motifs were identified among the binding sites of thechromatin-modulating RNAs roX2 and HOTAIR, indicating thatGA-enriched motifs may play an important role in the targeting of lncRNAs acrossthe genome19. Previously, several studies using differenttechniques have shown that GA-rich homopurine sequences can form triplexstructures383940. Overrepresentation of GA-rich sequencesamong the genomic binding sites of the lncRNAs analysed (MEG3,HOTAIR and roX) raises the possibility that the lncRNAs may berecruited to their target genes via RNA–DNA triplex formation. Byusing Triplexator software41 (which can predict triplex targetsites, TrTS), we found a greater number of the predicted TrTS in the MEG3peak summit (±200 bp from the centre of the peak) than theflanking sequences (200 bp upstream and 200 bp downstreamof the peak summit; Fig. 5b). Triplexator was also used toscan for triplex-forming oligonucleotides (TFOs) within the MEG3 RNA, andseveral TFOs with high scores were detected. Interestingly, the TFOs with highscores are also enriched with GA-rich sequences (Table3), indicating that the GA-rich sequences from target genes andMEG3 RNA could form triplex structures by forming Hoogsteen bondsbetween RNA and DNA. To test the ability of MEG3 lncRNA to form triplexstructures, we used a 20-nucleotide-long GA-rich RNA oligo (hereon referred toas MEG3 TFO) located at the 5′-end of the MEG3 RNA andits sequence overlap with the TFOs (TFO1, TFO2 and TFO3) with high score thatwere identified by Triplexator (Fig. 5c and Table 3). Using electrophoretic mobility shift assay, wetested the triplex-forming ability of the MEG3 TFO (single-stranded RNA,ssRNA) with the GA-rich (double-stranded DNA, dsDNA) MEG3 peak sequencesassociated with the selected TGF-β pathway target genes (TGFBR1,TGFB2 and SMAD2) in vitro (Fig.5d). Consistent with the Triplexator predictions, we observed a shiftin the end-labelled GA-rich dsDNA sequences when incubated with increasingconcentrations of the MEG3 TFO, indicating triplex formation between theMEG3 TFO and the GA-rich MEG3 peak summits (Fig. 5d, compare lane 1 with lanes 2 and 3), but not with a controlRNA oligo selected from the MEG3 lncRNA with no GA bias (Fig. 5d, compare lane 1 with lanes 8 and 9). The triplex structureswere sensitive to RNase A treatment but were resistant to RNase H digestion(Fig. 5d, lanes 4 and 5, respectively), while an invitro formed RNA–DNA hybrid was digested by RNase H (Supplementary Fig. 10a). Theseresults together suggest that the observed shift was not because ofWatson–Crick RNA–DNA pairing. We also observed that theseshifts were affected when specific competitor (the same GA-rich dsDNA oligo,unlabelled) was used but were unaffected by nonspecific competitor (unlabelledcontrol dsDNA oligo; Fig. 5d, compare lane 6 with 7). Wedid not observe any complex formation between MEG3 TFO incubated withend-labelled control DNA sequences (with no GA bias) or control RNA incubatedwith control DNA sequences (SupplementaryFig. 10b,c). To further check the specificity of the interactionbetween MEG3 TFO and GA-rich DNA sequences, we mutated the core sequencesof the TGFBR1-associated MEG3 peak and found that triplexformation was compromised between the mutant TGFBR1 dsDNA oligo and theMEG3 TFO (Fig. 5e). These observations suggestthat MEG3 may be recruited to genomic loci through the formation ofRNA–DNA triplex structures. We predicted the triplex-forming abilityof GA-rich motifs of another chromatin-interacting lncRNA, HOTAIR, andfound more Triplexator-predicted TrTS from the HOTAIR summit regions thanfrom the neighbouring sequences (Supplementary Fig. 10d,e).

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