Objective: Formation of DNA:DNA:RNA triplex through Hoogsteen base-pairing has been observed in artificial systems, but whether these interactions occur in cells and impact on cellular function is controversial. Moreover, the physiological relevance, the mechanistic mode of action and participating protein complexes of triplex forming lncRNAs are unclear. The long non-coding RNA (lncRNA) HIF1α-AS1 is located antisense to the important Hypoxia-inducible factor 1α gene, but the function is, however, unknown and was identified here.

Results: To identify functionally important DNA:DNA:RNA triplex-forming lncRNAs in human endothelial cells, we used a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies. The lncRNA HIF1α-AS1 was retrieved here as a top hit. Knockdown of the lncRNA in endothelial cells increased their angiogenic capacity. The lncRNA was reduced in endothelial cells isolated from glioblastoma and from lungs of patients with pulmonary arterial hypertension. In contrast, reoxygenation after hypoxia induced HIF1α-AS1. The lncRNA reduced the expression of numerous genes, including EPH Receptor A2 (EPHA2) and Adrenomedullin, through DNA:DNA:RNA triplex formation. Exchange of the triplex forming region of HIF1a-AS1 with other known triplex forming regions by CRISPR Arcitect abolished its effects on gene expression. Protein interaction studies revealed that HIF1α-AS1 interacts with the human silencing hub (HUSH) complex, which contains the epigenetic repressor MPP8. An assay for transposase-accessible chromatin followed by sequencing revealed that HIF1α-AS1 acts as an adapter for the HUSH complex on EPHA2 and Adrenomedullin.

Conclusions: As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is operative in vascular tissue to limit the angiogenic response and important for trans-acting gene expression control through the  recruitment of epigenetic silencer complexes.