MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures.

Tanmoy Mondal,Chandrasekhar Kanduri,Aristidis Moustakas,Andrew H Sims,Roshan Vaid,Stefan Enroth,Steven J.M Jones,Björn Reinius,Sanhita Mitra,Sireesha Uday,Alva Rani James,Arif Mohammed,Claes M Gustafsson,Emily Hoberg,Santhilal Subhash,Eduardo Gorab,Fredrik Westerlund,Ulf Gyllensten

doi:10.1038/ncomms8743

Tanmoy Mondal, Chandrasekhar Kanduri + Show 16 more

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https://doi.org/10.1038/ncomms8743

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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.

Highlights

We investigated the formation of RNA–DNA triplex structures by using an alternative method whereby biotin-labelled MEG3 TFO and a control RNA oligo were either used to transfect BT-549 cells (Fig. 5f) or incubated with nuclei isolated from BT-549 cells (Fig. 5g)
Upon pull-down with streptavidin magnetic beads, we found significant enrichment of the selected MEG3 peaks associated with the TGF-b genes—with MEG3 TFO compared with control oligo
We performed triplex-ChIP with anti-triplex dA.2rU antibody and observed enrichment of the selected MEG3 peaks associated with the TGFBR1, TGFB2 and SMAD2 genes

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Introduction

We investigated the formation of RNA–DNA triplex structures by using an alternative method whereby biotin-labelled MEG3 TFO and a control RNA oligo were either used to transfect BT-549 cells (Fig. 5f) or incubated with nuclei isolated from BT-549 cells (Fig. 5g). We analysed expression of the three key TGF-b genes TGFB2, TGFBR1 and SMAD2 in BT-549 cells after transfection with MEG3 TFO or control RNA oligo.

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