Limits...
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

Chromatin-binding sequences and PRC2-binding sequences of MEG3 lncRNAare functionally distinct.(a) MEG3-PRC2 in vitro binding assay. Left panel:schematic representation of WT MEG3, Δ46-56MEG3 andΔ345-348MEG3. Green and red boxes indicate PRC2- andchromatin-interacting sequences, respectively. Middle panel: bar diagramshowing the relative binding efficiency (as determined by RT–qPCR)of the sense WT MEG3, Δ46-56 MEG3 andΔ345-348 MEG3 RNAs in an in vitro PRC2-bindingassay. Binding assays with no PRC2 and antisense WT MEG3 served asnegative controls. The PRC2-binding efficiency of sense WT MEG3 wasset to 100, and the binding efficiency of the MEG3 mutants ispresented relative to WT MEG3 (±s.d.,n=3). Right panel: RT–qPCR showing thequantification of the input sense WT MEG3, MEG3 deletions(Δ340-348 or Δ345-348 MEG3) and antisense WT MEG3RNAs. (b) Deletion of MEG3 TFO leads to loss of chromatininteraction. Left panel: schematic display of interaction of the WTMEG3 and MEG3 mutants (Δ46-56MEG3 andΔ345-348MEG3) with the MEG3 peak sequences invivo. Red (MEG3 TFO) and green (PRC2-interacting region)colour-coded regions indicate the location of the deleted MEG3 RNAsequences 46-56 and 345-348, respectively. Biotin-labelled WT MEG3 orMEG3 mutants were used to transfect BT-549 cells followed bycrosslinking with formaldehyde. RNAse H-treated cell lysates were incubatedwith streptavidin beads to capture the MEG3 RNA-associated DNA.Middle panel: qPCR data are presented as the ratio of captured DNA in WTMEG3 or MEG3 mutants to captured non-biotinylatedMEG3 RNA (±s.d., n=3). Right panel:agarose gel picture showing the quality of the biotin-labelled WT and mutantMEG3 RNAs (500 ng of each biotin-RNA was loaded).(c) Model depicting how chromatin-interacting sequences ofMEG3 lncRNA-containing GA-rich sequences form RNA–DNAtriplex with the GA-rich DNA sequences to guide MEG3 lncRNA tochromatin. PRC2-interacting sequences of MEG3 lncRNA facilitaterecruitment of the PRC2 to distal regulatory elements, thereby establishingH3K27me3 marks to modulate gene expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4525211&req=5

f7: Chromatin-binding sequences and PRC2-binding sequences of MEG3 lncRNAare functionally distinct.(a) MEG3-PRC2 in vitro binding assay. Left panel:schematic representation of WT MEG3, Δ46-56MEG3 andΔ345-348MEG3. Green and red boxes indicate PRC2- andchromatin-interacting sequences, respectively. Middle panel: bar diagramshowing the relative binding efficiency (as determined by RT–qPCR)of the sense WT MEG3, Δ46-56 MEG3 andΔ345-348 MEG3 RNAs in an in vitro PRC2-bindingassay. Binding assays with no PRC2 and antisense WT MEG3 served asnegative controls. The PRC2-binding efficiency of sense WT MEG3 wasset to 100, and the binding efficiency of the MEG3 mutants ispresented relative to WT MEG3 (±s.d.,n=3). Right panel: RT–qPCR showing thequantification of the input sense WT MEG3, MEG3 deletions(Δ340-348 or Δ345-348 MEG3) and antisense WT MEG3RNAs. (b) Deletion of MEG3 TFO leads to loss of chromatininteraction. Left panel: schematic display of interaction of the WTMEG3 and MEG3 mutants (Δ46-56MEG3 andΔ345-348MEG3) with the MEG3 peak sequences invivo. Red (MEG3 TFO) and green (PRC2-interacting region)colour-coded regions indicate the location of the deleted MEG3 RNAsequences 46-56 and 345-348, respectively. Biotin-labelled WT MEG3 orMEG3 mutants were used to transfect BT-549 cells followed bycrosslinking with formaldehyde. RNAse H-treated cell lysates were incubatedwith streptavidin beads to capture the MEG3 RNA-associated DNA.Middle panel: qPCR data are presented as the ratio of captured DNA in WTMEG3 or MEG3 mutants to captured non-biotinylatedMEG3 RNA (±s.d., n=3). Right panel:agarose gel picture showing the quality of the biotin-labelled WT and mutantMEG3 RNAs (500 ng of each biotin-RNA was loaded).(c) Model depicting how chromatin-interacting sequences ofMEG3 lncRNA-containing GA-rich sequences form RNA–DNAtriplex with the GA-rich DNA sequences to guide MEG3 lncRNA tochromatin. PRC2-interacting sequences of MEG3 lncRNA facilitaterecruitment of the PRC2 to distal regulatory elements, thereby establishingH3K27me3 marks to modulate gene expression.

Mentions: We wanted to determine whether the MEG3 RNA sequences required for thePRC2 interaction and RNA–DNA triplex formation are functionallydistinct. For this purpose, we generated a MEG3 mutant by deleting thecore MEG3 TFO (the TFO used in the triplex assay above) containingGA-rich sequences and named this Δ46-56 MEG3 (46–56indicates the position of the nucleotides with respect to the 5′-endof MEG3). We found that the TFO deletion had no effect on the interactionof PRC2 with MEG3 (Fig. 7a). Considering thatΔ345-348 MEG3 affects the PRC2 interaction of WT MEG3(Figs 7a and 2e), we decided totest the chromatin-interacting property of the WT and MEG3 RNAs withdeletions (Δ46-56 MEG3 and Δ345-348 MEG3) bytransfecting BT-549 cells with in vitro-synthesized biotin-labelled WT ormutant MEG3 RNAs (Δ46-56 MEG3 or Δ345-348MEG3). We found that the WT and Δ345-348 MEG3 RNAscould pull-down MEG3 peak sequences associated with theTGF-β genes (TGFB2, TGFBR1 and SMAD2),but not when the pull-down was performed with the Δ46-56 MEG3TFO deletion (Fig. 7b). This suggests that the decrease inthe association between MEG3 and PRC2 does not have any effect on thechromatin-binding property of MEG3 RNA. Taken together, theseobservations indicate that the chromatin targeting and PRC2 interactionproperties of MEG3 lncRNA are mediated by distinct RNA sequences (Fig. 7c).


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)

Chromatin-binding sequences and PRC2-binding sequences of MEG3 lncRNAare functionally distinct.(a) MEG3-PRC2 in vitro binding assay. Left panel:schematic representation of WT MEG3, Δ46-56MEG3 andΔ345-348MEG3. Green and red boxes indicate PRC2- andchromatin-interacting sequences, respectively. Middle panel: bar diagramshowing the relative binding efficiency (as determined by RT–qPCR)of the sense WT MEG3, Δ46-56 MEG3 andΔ345-348 MEG3 RNAs in an in vitro PRC2-bindingassay. Binding assays with no PRC2 and antisense WT MEG3 served asnegative controls. The PRC2-binding efficiency of sense WT MEG3 wasset to 100, and the binding efficiency of the MEG3 mutants ispresented relative to WT MEG3 (±s.d.,n=3). Right panel: RT–qPCR showing thequantification of the input sense WT MEG3, MEG3 deletions(Δ340-348 or Δ345-348 MEG3) and antisense WT MEG3RNAs. (b) Deletion of MEG3 TFO leads to loss of chromatininteraction. Left panel: schematic display of interaction of the WTMEG3 and MEG3 mutants (Δ46-56MEG3 andΔ345-348MEG3) with the MEG3 peak sequences invivo. Red (MEG3 TFO) and green (PRC2-interacting region)colour-coded regions indicate the location of the deleted MEG3 RNAsequences 46-56 and 345-348, respectively. Biotin-labelled WT MEG3 orMEG3 mutants were used to transfect BT-549 cells followed bycrosslinking with formaldehyde. RNAse H-treated cell lysates were incubatedwith streptavidin beads to capture the MEG3 RNA-associated DNA.Middle panel: qPCR data are presented as the ratio of captured DNA in WTMEG3 or MEG3 mutants to captured non-biotinylatedMEG3 RNA (±s.d., n=3). Right panel:agarose gel picture showing the quality of the biotin-labelled WT and mutantMEG3 RNAs (500 ng of each biotin-RNA was loaded).(c) Model depicting how chromatin-interacting sequences ofMEG3 lncRNA-containing GA-rich sequences form RNA–DNAtriplex with the GA-rich DNA sequences to guide MEG3 lncRNA tochromatin. PRC2-interacting sequences of MEG3 lncRNA facilitaterecruitment of the PRC2 to distal regulatory elements, thereby establishingH3K27me3 marks to modulate gene expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525211&req=5

f7: Chromatin-binding sequences and PRC2-binding sequences of MEG3 lncRNAare functionally distinct.(a) MEG3-PRC2 in vitro binding assay. Left panel:schematic representation of WT MEG3, Δ46-56MEG3 andΔ345-348MEG3. Green and red boxes indicate PRC2- andchromatin-interacting sequences, respectively. Middle panel: bar diagramshowing the relative binding efficiency (as determined by RT–qPCR)of the sense WT MEG3, Δ46-56 MEG3 andΔ345-348 MEG3 RNAs in an in vitro PRC2-bindingassay. Binding assays with no PRC2 and antisense WT MEG3 served asnegative controls. The PRC2-binding efficiency of sense WT MEG3 wasset to 100, and the binding efficiency of the MEG3 mutants ispresented relative to WT MEG3 (±s.d.,n=3). Right panel: RT–qPCR showing thequantification of the input sense WT MEG3, MEG3 deletions(Δ340-348 or Δ345-348 MEG3) and antisense WT MEG3RNAs. (b) Deletion of MEG3 TFO leads to loss of chromatininteraction. Left panel: schematic display of interaction of the WTMEG3 and MEG3 mutants (Δ46-56MEG3 andΔ345-348MEG3) with the MEG3 peak sequences invivo. Red (MEG3 TFO) and green (PRC2-interacting region)colour-coded regions indicate the location of the deleted MEG3 RNAsequences 46-56 and 345-348, respectively. Biotin-labelled WT MEG3 orMEG3 mutants were used to transfect BT-549 cells followed bycrosslinking with formaldehyde. RNAse H-treated cell lysates were incubatedwith streptavidin beads to capture the MEG3 RNA-associated DNA.Middle panel: qPCR data are presented as the ratio of captured DNA in WTMEG3 or MEG3 mutants to captured non-biotinylatedMEG3 RNA (±s.d., n=3). Right panel:agarose gel picture showing the quality of the biotin-labelled WT and mutantMEG3 RNAs (500 ng of each biotin-RNA was loaded).(c) Model depicting how chromatin-interacting sequences ofMEG3 lncRNA-containing GA-rich sequences form RNA–DNAtriplex with the GA-rich DNA sequences to guide MEG3 lncRNA tochromatin. PRC2-interacting sequences of MEG3 lncRNA facilitaterecruitment of the PRC2 to distal regulatory elements, thereby establishingH3K27me3 marks to modulate gene expression.
Mentions: We wanted to determine whether the MEG3 RNA sequences required for thePRC2 interaction and RNA–DNA triplex formation are functionallydistinct. For this purpose, we generated a MEG3 mutant by deleting thecore MEG3 TFO (the TFO used in the triplex assay above) containingGA-rich sequences and named this Δ46-56 MEG3 (46–56indicates the position of the nucleotides with respect to the 5′-endof MEG3). We found that the TFO deletion had no effect on the interactionof PRC2 with MEG3 (Fig. 7a). Considering thatΔ345-348 MEG3 affects the PRC2 interaction of WT MEG3(Figs 7a and 2e), we decided totest the chromatin-interacting property of the WT and MEG3 RNAs withdeletions (Δ46-56 MEG3 and Δ345-348 MEG3) bytransfecting BT-549 cells with in vitro-synthesized biotin-labelled WT ormutant MEG3 RNAs (Δ46-56 MEG3 or Δ345-348MEG3). We found that the WT and Δ345-348 MEG3 RNAscould pull-down MEG3 peak sequences associated with theTGF-β genes (TGFB2, TGFBR1 and SMAD2),but not when the pull-down was performed with the Δ46-56 MEG3TFO deletion (Fig. 7b). This suggests that the decrease inthe association between MEG3 and PRC2 does not have any effect on thechromatin-binding property of MEG3 RNA. Taken together, theseobservations indicate that the chromatin targeting and PRC2 interactionproperties of MEG3 lncRNA are mediated by distinct RNA sequences (Fig. 7c).

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