HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex.

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.

Detection of RNA-DNA binding sites in long noncoding RNAs.

Long non-coding RNAs (lncRNAs) can act as scaffolds that promote the interaction of proteins, RNA, and DNA. There is increasing evidence of sequence-specific interactions of lncRNAs with DNA via triple-helix (triplex) formation. This process allows lncRNAs to recruit protein complexes to specific genomic regions and regulate gene expression. Here we propose a computational method called Triplex Domain Finder (TDF) to detect triplexes and characterize DNA-binding domains and DNA targets statistically. Case studies showed that this approach can detect the known domains of lncRNAs Fendrr, HOTAIR and MEG3. Moreover, we validated a novel DNA-binding domain in MEG3 by a genome-wide sequencing method. We used TDF to perform a systematic analysis of the triplex-forming potential of lncRNAs relevant to human cardiac differentiation. We demonstrated that the lncRNA with the highest triplex-forming potential, GATA6-AS, forms triple helices in the promoter of genes relevant to cardiac development. Moreover, down-regulation of GATA6-AS impairs GATA6 expression and cardiac development. These data indicate the unique ability of our computational tool to identify novel triplex-forming lncRNAs and their target genes.

Isolation and genome-wide characterization of cellular DNA:RNA triplex structures.

RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple helical structure that anchors the RNA to specific sequences and allows guiding of transcription regulators to distinct genomic loci. To unravel the prevalence of DNA:RNA triplexes in living cells, we have established a fast and cost-effective method that allows genome-wide mapping of DNA:RNA triplex interactions. In contrast to previous approaches applied for the identification of chromatin-associated RNAs, this method uses protein-free nucleic acids isolated from chromatin. High-throughput sequencing and computational analysis of DNA-associated RNA revealed a large set of RNAs which originate from non-coding and coding loci, including super-enhancers and repeat elements. Combined analysis of DNA-associated RNA and RNA-associated DNA identified genomic DNA:RNA triplex structures. The results suggest that triplex formation is a general mechanism of RNA-mediated target-site recognition, which has major impact on biological functions.