The lncRNA HOTAIR impacts on mesenchymal stem cells via triple helix formation

Abstract

There is a growing perception that long non-coding RNAs (lncRNAs) modulate cellular function. In this study, we 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. 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. Our 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.

Figure 1.

HOTAIR binding is enriched close to senescence-associated hypermethylation. (A) DNA-methylation was measured in MSCs and iPSCs with Infinium Human Methylation450K BeadChips (n = 4) (8). These DNAm profiles revealed higher methylation levels in the HOTAIR locus in MSCs of later passage as compared to early passage, particularly at two CpGs (*P < 0.05; adjusted paired limma t-test; cg14691529 and cg06850442). (B) HOTAIR and ANRIL expression were further analyzed by qRT-PCR in MSCs of early (P4) and late (P13) passage (n = 3; normalized to MSC P4; *P < 0.05). (C) Senescence-associated DNAm changes in MSCs (hypo- and hypermethylated CpGs) were correlated with HOTAIR binding regions of a publically available ChIRP-sequencing dataset of MDA-MB-231 breast cancer cells (48). This analysis indicated that HOTAIR binding was significantly enriched in the vicinity of CpGs that are hypermethylated upon culture expansion in comparison to random selected CpGs (red line indicates P = 0.05 cut-off).

Figure 2.

HOTAIR overexpression reduces adipogenic differentiation. (A) Retroviral overexpression of HOTAIR in MSCs of passage 4 revealed 97-fold upregulation by qRT-PCR analysis two weeks after transfection (GFP-insert was used for control; normalized to untreated control; n = 7). (B) Proliferation within 5 days (as determined by cell counting in a Neubauer cell chamber) was not significantly affected by overexpression (n = 5). (C and D) Osteogenic differentiation was not significantly affected by HOTAIR overexpression (Alizarin red extraction with acetic acid after 21 days; absorption measured on a plate reader; n = 11). (E and F) Adipogenic differentiation was reduced upon HOTAIR overexpression (percentage of BODIPY positive cells as compared to all nuclei counterstained with DAPI; n = 9). Paired t-test: *P < 0.05; **P < 0.01; Size bar = 100 μm.

Figure 3.

HOTAIR knockdown impairs proliferation and enhances in vitro differentiation. (A) Quantitative RT-PCR validation of siRNA mediated HOTAIR knockdown in MSCs after 48 h (normalized to controls with non-specific siRNA with Alexa Fluor (siAF); n = 9). (B) Proliferation of MSCs, as determined by cell counting after 5 days, was significantly reduced by HOTAIR knockdown (n = 5). In vitro differentiation towards osteogenic (C and D) and adipogenic (E and F) lineages was analyzed after 24 days (with repeated siRNA transfections at days 1, 8, 15 and 22). Alizarin red analysis indicated increased osteogenic differentiation upon HOTAIR knockdown (n = 6). Furthermore, the percentage of BODIPY-positive cells with fat droplets increased upon HOTAIR knockdown (n = 5). (G) HOTAIR expression in MSCs was significantly reduced upon differentiation of MSCs towards adipogenic lineage (n = 3; normalized to untreated MSCs). Paired t-test: *P < 0.05; **P < 0.01; size bar = 100 μm.

Figure 4.

HOTAIR modulates gene expression patterns and DNA methylation. Scatter plots demonstrate gene expression changes of MSCs upon either HOTAIR overexpression (A) or knockdown (B) in comparison to corresponding controls (means of three replicas). Genes with moderate upregulation and downregulation (log fold-change > 1.2) are indicated in green and red, respectively. HOTAIR revealed highest upregulation in overexpression data but was only moderately downregulated upon siRNA knockdown (indicated by arrows). (C) Genes that were either upregulated (900 genes; green) or downregulated (1042 genes, red) upon HOTAIR overexpression were analyzed in RNA-seq data of replicative senescent MSCs. Downregulated genes were significantly higher expressed at later passage (P = 1.7 × 10⁻⁵). (D) In contrast, genes that were either upregulated (794; red) or downregulated (542; green) upon HOTAIR knockdown revealed no significant changes in expression during replicative senescence. DNA methylation profiles of MSCs (P4) were analyzed upon HOTAIR overexpression or knockdown with Infinium Human Methylation450K BeadChips (Illumina, San Diego, USA). Scatter plots reveal DNAm changes upon (E) overexpression (compared to GFP control) and (F) knockdown (compared to fluorescence labelled negative antisense control; siAF). CpGs with >10% hypermethylation (green) and hypomethylation (red) are highlighted. (G) CpGs that were hypomethylated upon HOTAIR overexpression (708 CpGs) were overall significantly higher methylated in early versus later passages of MSCs (P = 1.6 × 10⁻⁹) (8). (H) CpGs with differential DNAm upon HOTAIR knockdown did not reveal a clear trend with senescence-associated DNAm.

Figure 5.

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.

Figure 6.

Electrophoretic mobility shift assays support triple helix formation. Computationally predicted triple helix forming sites in the promoter of (A) PCDH7 and (B) HOXB2 that were considered for in vitro validation. (C and D) Electrophoretic mobility shift assay of predicted binding domains (PCDH7 and HOXB2). Complementary oligodeoxynucleotides were preincubated to form double stranded DNA and then incubated with either specific RNA of predicted triplex binding domain II in HOTAIR, reverse RNA, or non-specific control RNA (the corresponding region of the control RNA is indicated in Figure 5B; notably, this RNA was also detected by Gel Red staining possibly due to hairpin formation; RNA was applied in 2-fold, 10-fold, 25-fold or 50-fold excess; 1.1 equivalents were used of the pyrimidine-rich DNA strand to reduce the possibility of DNA:DNA-DNA triplex formation. A mobility shift that indicates triplex formation was only observed with the specific sequences of HOTAIR domain II in both predicted target regions. (E) Triplex formation was then analyzed using different concentrations of RNA. (F) To rule out that conventional Watson-Crick hybridization between RNA and DNA results in mobility shift we treated the complexes with RNaseH. As expected, RNA in triplexes was protected from digestion, whereas it was digested in RNA:DNA double helices.