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

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

PROVIDER: PRJEB7307 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR652839.fastq.gz	Fastqsanger.gz
	ERR652840.fastq.gz	Fastqsanger.gz
	ERR652841.fastq.gz	Fastqsanger.gz
	ERR652842.fastq.gz	Fastqsanger.gz
	ERR652843.fastq.gz	Fastqsanger.gz

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

Project description: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.

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Detection of RNA–DNA binding sites in long noncoding RNAs

Project description:Triple helix is a potential mechanism how lncRNAs interact with the genome. Our in silico method (Triplex Domain Finder) is able to predict the binding sites of lncRNA MEG3 and GATA6-AS in the genome. In order to validate these predictions, we develop DBD-Capture-Seq to capture the DNA loci where the given RNA oligo binds to via triplex. Method: Different RNA oligos are used The biotinylated RNA oligos (MEG3 TFR1, MEG3 TFR2, and GATA6-AS TFR1) were incubated with sheared genomic DNA to allow for triplex formation. After binding to streptavidin-coated beads, RNA-associated DNA was eluted and subjected to deep sequencing. Control experiments were conducted in the absence of biotinylated RNA oligos.

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Project description:Triplex DNA structures, formed by a third DNA strand wrapped around the major groove of double helix, are key molecular regulators and genomic threats that are pharmacologically exploitable. However, the regulatory network governing triplex DNA dynamics are poorly understood. Here we performed chemoproteomic profiling of triplex DNA interactome in live cells to address this knowledge gap. We developed and validated a chemical probe that exhibits exceptional specificity for recognizing triplex DNA structures. By employing a co-binding-mediated proximity capture strategy, we enriched triplex DNA interactome for quantitative proteomics analysis. This enabled the identification of a comprehensive list of triplex DNA interacting proteins, characterized by diverse binding properties and regulatory mechanisms in native chromatin context. As a demonstration, we further validated DDX3X as the first ATP-independent helicase capable of resolving triplex DNA structures with 5' overhangs on the third DNA strand to prevent triplex-DNA-induced DNA damages. Overall, our triplex DNA interactome offers a valuable resource for investigating the biology of triplex DNA in both health and disease.

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