There is a growing perception that long non-coding RNAs (lncRNAs) modulate cellular function. In this study, researchers from RWTH University Medical School analyzed the role of the lncRNA HOTAIR in mesenchymal stem cells (MSCs) with particular focus on senescence-associated changes in gene expression and DNA-methylation (DNAm). HOTAIR binding sites were enriched at genomic regions that become hypermethylated with increasing cell culture passage. Overexpression and knockdown of HOTAIR inhibited or stimulated adipogenic differentiation of MSCs, respectively. Modification of HOTAIR expression evoked only very moderate effects on gene expression, particularly of polycomb group target genes. Furthermore, overexpression and knockdown of HOTAIR resulted in DNAm changes at HOTAIR binding sites.
To find functionally enriched triple helix forming domains in HOTAIR, the researchers used Triplex Domain Finder (TDF; http://www.regulatory-genomics.org/tdf/). Five potential triple helix forming domains were predicted within the HOTAIR sequence based on reverse Hoogsteen hydrogen bonds. Notably, the predicted triple helix target sites for these HOTAIR domains were also enriched in differentially expressed genes and close to DNAm changes upon modulation of HOTAIR. Electrophoretic mobility shift assays provided further evidence that HOTAIR domains form RNA–DNA–DNA triplexes with predicted target sites. These results demonstrate that HOTAIR impacts on differentiation of MSCs and that it is associated with senescence-associated DNAm. Targeting of epigenetic modifiers to relevant loci in the genome may involve triple helix formation with HOTAIR.
Triple Helix forming potential of the HOTAIR sequence
(A) RNA might bind to a DNA double helix through reverse Hoogsteen hydrogen bonds and form a triple helix. (B) ChIRP-Seq-data of HOTAIR and the HOTAIR sequence were used for analysis with Triplex Domain Finder; Red peaks show significant DNA binding domains within the HOTAIR-sequence (P < 0.05); yellow boxes mark regions of RNA interaction with PRC2 and LSD1. (C) Number of triple helix binding sites for each significant DNA binding domain on ChIRP-Seq and random regions.