REpressive chromatin lncRNA
Characterization of repressive chromatin associated lncRNAs
Identification of repressive chromatin-associated lncRNAs using ChRIP-seq. a) The pie chart shows 276 lncRNAs enriched in both EZH2 and H3K27me3 ChRIP-seq samples compared with the nuclear RNA (input). The P value was obtained by performing a hypergeometric test using all the lncRNAs in our analysis. b) All the possible conversions present in the EZH2 ChRIP-seq sample. T-to-C conversion and the reverse-strand A-to-G conversions were predominant among all the possible conversion events. d) LncRNAs (1,046; annotated and non-annotated) harbour EZH2-specific (17,652) T-to-C conversion site. Seventy repressive chromatin-enriched lncRNAs (out of 276) carry T-to-C transitions, including known PRC2-interacting lncRNAs such as MEG3, KCNQ1OT1 and BDNF-AS1.The P value was obtained by performing a hypergeometric test using all the lncRNAs considered in our analysis. b) The distribution of the sequencing reads on MEG3 transcript from H3K27me3, EZH2-enriched chromatin fractions and input RNA samples. The Y-axis depicts the RPKM (Reads per kilobase per million) for MEG3 in H3K27me3, EZH2 ChRIP RNA and input RNA samples. The fold enrichment (FC) in H3K27me3 and EZH2 ChRIP RNA compared with input is indicated.
Pathways regulated by repressive chromatin associated lncRNA MEG3 and EZH2 (PRC2 catalytic subunit)
MEG3 and EZH2 share common gene targets. a) Venn diagram showing the overlap of the deregulated protein-coding genes identified by microarray and RNA sequencing after downregulation of MEG3 and EZH2 using siRNA in BT-549 cells. The P values were obtained by performing a hypergeometric test using all protein-coding genes as a background. b) Pathway analysis of the overlapped genes from microarray and RNA-seq after MEG3 and EZH2 downregulation in BT-549 cells, using KEGG annotation. c) Venn diagram showing the number of genes deregulated after downregulation of MEG3 and EZH2 using siRNA in BT-549 cells, and the degree of overlap between the MEG3- and EZH2-dependent genes. The P values were obtained by hypergeometric test using all protein-coding genes as a background.
MEG3 forming RNA-DNA triplex structure and recruits PRC2 to the distal regulatory elements to control expression of TGF-beta pathway genes
a) Summary of MEG3 peaks with associated genes and their overlap with H3K4me1 peaks in BT-549 cells. b) Predicted GA-rich motifs enriched in all MEG3 peaks and peaks associated with deregulated genes using MEME-ChIP (right panel). Number of TrTS over the MEG3 peak summits and neighbouring regions, predicted by Triplexator (left panel). c) Summary of predicted Triplex Forming Oligos (TFOs). d) Model depicting how chromatin-interacting sequences of MEG3 lncRNA-containing GA-rich sequences form RNA–DNA triplex with the GA-rich DNA sequences to guide MEG3 lncRNA to chromatin. PRC2-interacting sequences of MEG3 lncRNA facilitate recruitment of the PRC2 to distal regulatory elements, thereby establishing H3K27me3 marks to modulate gene expression.