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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/EZH2 functional interaction regulates TGF-βpathway genes.(a–c) MEG3 and EZH2 share common gene targets.(a) Venn diagram showing the number of genes deregulated afterdownregulation of MEG3 and EZH2 using siRNA in BT-549 and HFcells, and the degree of overlap between the MEG3- and EZH2-dependentgenes. The P values were obtained by hypergeometric test using allprotein-coding genes as a background. (b) EZH2 protein levels, asdetermined by western blotting, following EZH2 and MEG3downregulation in BT-549 and HF cells. Tubulin was used as a loadingcontrol. (c) RT–qPCR analysis of EZH2 andMEG3 mRNA expression in Ctrlsi, EZH2si and MEG3sitransfected BT-549 and HF cells (±s.d., n=3).(d) RT–qPCR analysis of TGFB2, TGFBR1 andSMAD2 gene expression in Ctrlsi, MEG3si and EZH2sitransfected BT-549 cells (±s.d., n=3).(e) Immunoblot showing SMAD2, TGFBR1 and tubulin protein levelsfollowing transfection of BT-549 cells with Ctrlsi and MEG3si.(f) Bar graph showing RT–qPCR analysis of TGFB2,TGFBR1 and SMAD2 mRNA levels after overexpression ofMEG3 (pREP4MEG3) in BT-549 and MDA-MB-231 cells. Thelevels in pREP4MEG3 are presented relative to CtrlpREP4(±s.d., n=3). EZH2 was used as a controlshowing no change in expression after overexpression of MEG3. TheP values were calculated using Student's t-test(two-tailed, two-sample unequal variance), *P<0.05.(g) Downregulation of MEG3 influences the invasiveproperty of BT-549 cells through regulation of the TGF-β pathway.Images showing Matrigel invasion of the BT-549 cells. The two images in theupper panel show invasion of BT-549 cells infected with Ctrlsh lentivirus,and Ctrlsh infection followed by incubation with TGF-β2 ligand(Ctrlsh-TGFB2). The images in the bottom panel show the cells infected withMEG3Sh and MEG3sh infection followed by incubation withTGF-β inhibitor (MEG3sh+TGF-βin).Scale bar, 10 μm. The bar graph shows quantification(±s.d., n=3) of the matrix invaded cells inMEG3sh relative to the Ctrlsh. The P values werecalculated using Student's t-test (two-tailed, two-sampleunequal variance), *P<0.05,**P<0.01.
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f3: MEG3/EZH2 functional interaction regulates TGF-βpathway genes.(a–c) MEG3 and EZH2 share common gene targets.(a) Venn diagram showing the number of genes deregulated afterdownregulation of MEG3 and EZH2 using siRNA in BT-549 and HFcells, and the degree of overlap between the MEG3- and EZH2-dependentgenes. The P values were obtained by hypergeometric test using allprotein-coding genes as a background. (b) EZH2 protein levels, asdetermined by western blotting, following EZH2 and MEG3downregulation in BT-549 and HF cells. Tubulin was used as a loadingcontrol. (c) RT–qPCR analysis of EZH2 andMEG3 mRNA expression in Ctrlsi, EZH2si and MEG3sitransfected BT-549 and HF cells (±s.d., n=3).(d) RT–qPCR analysis of TGFB2, TGFBR1 andSMAD2 gene expression in Ctrlsi, MEG3si and EZH2sitransfected BT-549 cells (±s.d., n=3).(e) Immunoblot showing SMAD2, TGFBR1 and tubulin protein levelsfollowing transfection of BT-549 cells with Ctrlsi and MEG3si.(f) Bar graph showing RT–qPCR analysis of TGFB2,TGFBR1 and SMAD2 mRNA levels after overexpression ofMEG3 (pREP4MEG3) in BT-549 and MDA-MB-231 cells. Thelevels in pREP4MEG3 are presented relative to CtrlpREP4(±s.d., n=3). EZH2 was used as a controlshowing no change in expression after overexpression of MEG3. TheP values were calculated using Student's t-test(two-tailed, two-sample unequal variance), *P<0.05.(g) Downregulation of MEG3 influences the invasiveproperty of BT-549 cells through regulation of the TGF-β pathway.Images showing Matrigel invasion of the BT-549 cells. The two images in theupper panel show invasion of BT-549 cells infected with Ctrlsh lentivirus,and Ctrlsh infection followed by incubation with TGF-β2 ligand(Ctrlsh-TGFB2). The images in the bottom panel show the cells infected withMEG3Sh and MEG3sh infection followed by incubation withTGF-β inhibitor (MEG3sh+TGF-βin).Scale bar, 10 μm. The bar graph shows quantification(±s.d., n=3) of the matrix invaded cells inMEG3sh relative to the Ctrlsh. The P values werecalculated using Student's t-test (two-tailed, two-sampleunequal variance), *P<0.05,**P<0.01.

Mentions: To gain more insights into the functional significance of the interaction of PRC2with the MEG3 lncRNA, both EZH2 and MEG3 transcripts weredownregulated in BT-549 and HF cells using small interfering RNA (siRNA), andgene expression profiles were measured using microarray. We observed asignificant overlap among the deregulated genes between the EZH2 andMEG3 data sets from BT-549 and HF cells, indicating a functionalassociation between the MEG3 lncRNA and EZH2 (Fig.3a, Supplementary Data 5and Supplementary Data 6).MEG3 downregulation did not interfere with EZH2 RNA andprotein levels, and similarly, EZH2 downregulation did not affectMEG3 transcript levels (Fig. 3b,c), suggestingthat the overlap observed between the deregulated genes in the EZH2 andMEG3 data sets was not due to changes in EZH2 levels uponMEG3 downregulation or in MEG3 levels upon EZH2downregulation. To rule out off-target effects of the MEG3 siRNA, we usedreverse transcription (RT)–qPCR to validate four target genes uponMEG3 downregulation using an alternative siRNA against MEG3(Supplementary Fig. 3b,c).


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/EZH2 functional interaction regulates TGF-βpathway genes.(a–c) MEG3 and EZH2 share common gene targets.(a) Venn diagram showing the number of genes deregulated afterdownregulation of MEG3 and EZH2 using siRNA in BT-549 and HFcells, and the degree of overlap between the MEG3- and EZH2-dependentgenes. The P values were obtained by hypergeometric test using allprotein-coding genes as a background. (b) EZH2 protein levels, asdetermined by western blotting, following EZH2 and MEG3downregulation in BT-549 and HF cells. Tubulin was used as a loadingcontrol. (c) RT–qPCR analysis of EZH2 andMEG3 mRNA expression in Ctrlsi, EZH2si and MEG3sitransfected BT-549 and HF cells (±s.d., n=3).(d) RT–qPCR analysis of TGFB2, TGFBR1 andSMAD2 gene expression in Ctrlsi, MEG3si and EZH2sitransfected BT-549 cells (±s.d., n=3).(e) Immunoblot showing SMAD2, TGFBR1 and tubulin protein levelsfollowing transfection of BT-549 cells with Ctrlsi and MEG3si.(f) Bar graph showing RT–qPCR analysis of TGFB2,TGFBR1 and SMAD2 mRNA levels after overexpression ofMEG3 (pREP4MEG3) in BT-549 and MDA-MB-231 cells. Thelevels in pREP4MEG3 are presented relative to CtrlpREP4(±s.d., n=3). EZH2 was used as a controlshowing no change in expression after overexpression of MEG3. TheP values were calculated using Student's t-test(two-tailed, two-sample unequal variance), *P<0.05.(g) Downregulation of MEG3 influences the invasiveproperty of BT-549 cells through regulation of the TGF-β pathway.Images showing Matrigel invasion of the BT-549 cells. The two images in theupper panel show invasion of BT-549 cells infected with Ctrlsh lentivirus,and Ctrlsh infection followed by incubation with TGF-β2 ligand(Ctrlsh-TGFB2). The images in the bottom panel show the cells infected withMEG3Sh and MEG3sh infection followed by incubation withTGF-β inhibitor (MEG3sh+TGF-βin).Scale bar, 10 μm. The bar graph shows quantification(±s.d., n=3) of the matrix invaded cells inMEG3sh relative to the Ctrlsh. The P values werecalculated using Student's t-test (two-tailed, two-sampleunequal variance), *P<0.05,**P<0.01.
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Related In: Results  -  Collection

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f3: MEG3/EZH2 functional interaction regulates TGF-βpathway genes.(a–c) MEG3 and EZH2 share common gene targets.(a) Venn diagram showing the number of genes deregulated afterdownregulation of MEG3 and EZH2 using siRNA in BT-549 and HFcells, and the degree of overlap between the MEG3- and EZH2-dependentgenes. The P values were obtained by hypergeometric test using allprotein-coding genes as a background. (b) EZH2 protein levels, asdetermined by western blotting, following EZH2 and MEG3downregulation in BT-549 and HF cells. Tubulin was used as a loadingcontrol. (c) RT–qPCR analysis of EZH2 andMEG3 mRNA expression in Ctrlsi, EZH2si and MEG3sitransfected BT-549 and HF cells (±s.d., n=3).(d) RT–qPCR analysis of TGFB2, TGFBR1 andSMAD2 gene expression in Ctrlsi, MEG3si and EZH2sitransfected BT-549 cells (±s.d., n=3).(e) Immunoblot showing SMAD2, TGFBR1 and tubulin protein levelsfollowing transfection of BT-549 cells with Ctrlsi and MEG3si.(f) Bar graph showing RT–qPCR analysis of TGFB2,TGFBR1 and SMAD2 mRNA levels after overexpression ofMEG3 (pREP4MEG3) in BT-549 and MDA-MB-231 cells. Thelevels in pREP4MEG3 are presented relative to CtrlpREP4(±s.d., n=3). EZH2 was used as a controlshowing no change in expression after overexpression of MEG3. TheP values were calculated using Student's t-test(two-tailed, two-sample unequal variance), *P<0.05.(g) Downregulation of MEG3 influences the invasiveproperty of BT-549 cells through regulation of the TGF-β pathway.Images showing Matrigel invasion of the BT-549 cells. The two images in theupper panel show invasion of BT-549 cells infected with Ctrlsh lentivirus,and Ctrlsh infection followed by incubation with TGF-β2 ligand(Ctrlsh-TGFB2). The images in the bottom panel show the cells infected withMEG3Sh and MEG3sh infection followed by incubation withTGF-β inhibitor (MEG3sh+TGF-βin).Scale bar, 10 μm. The bar graph shows quantification(±s.d., n=3) of the matrix invaded cells inMEG3sh relative to the Ctrlsh. The P values werecalculated using Student's t-test (two-tailed, two-sampleunequal variance), *P<0.05,**P<0.01.
Mentions: To gain more insights into the functional significance of the interaction of PRC2with the MEG3 lncRNA, both EZH2 and MEG3 transcripts weredownregulated in BT-549 and HF cells using small interfering RNA (siRNA), andgene expression profiles were measured using microarray. We observed asignificant overlap among the deregulated genes between the EZH2 andMEG3 data sets from BT-549 and HF cells, indicating a functionalassociation between the MEG3 lncRNA and EZH2 (Fig.3a, Supplementary Data 5and Supplementary Data 6).MEG3 downregulation did not interfere with EZH2 RNA andprotein levels, and similarly, EZH2 downregulation did not affectMEG3 transcript levels (Fig. 3b,c), suggestingthat the overlap observed between the deregulated genes in the EZH2 andMEG3 data sets was not due to changes in EZH2 levels uponMEG3 downregulation or in MEG3 levels upon EZH2downregulation. To rule out off-target effects of the MEG3 siRNA, we usedreverse transcription (RT)–qPCR to validate four target genes uponMEG3 downregulation using an alternative siRNA against MEG3(Supplementary Fig. 3b,c).

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