The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

Abstract

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT.

Figure 1

HOTAIR expression correlates to mesenchymal genes. (a) RT–qPCR analysis for HOTAIR, EZH2, the indicated epithelial (E-cadherin and HNF4α) and mesenchymal (Snail, fibronectin and vimentin) genes on hepatocytes treated with TGFβ or not treated (NT) with TGFβ for 24 h. The values are calculated by the 2^(−ΔCt) method, expressed as fold of expression versus the control (arbitrary value=1) and shown as means±s.e.m. Statistically significant differences are reported (*P<0.05, **P<0.01) for five independent experiments (NS=no significance). (b) RT–qPCR analysis for the same genes as in (a) in hepatocytes overexpressing HOTAIR (HOTAIR) or an empty vector (Ctr). The values are calculated by the 2^(−ΔCt) method, expressed as fold of expression versus the control (arbitrary value=1) and shown as means±s.e.m. No statistically significant differences were evaluated for five independent experiments.

Figure 2

HOTAIR has a functional role in EMT. (a) RT–qPCR analysis for the indicated markers on HOTAIR-silenced hepatocytes (siHotair), compared with control siGFP cells (siCtr), treated (TGFβ) or not (Ctr) with TGFβ for 24 h. The values are calculated by the 2^(−ΔCt) method, expressed as fold of expression versus the control (arbitrary value=1) and shown as means±s.e.m. Statistically significant differences are reported (*P<0.05, **P<0.01) for five independent experiments (NS=no significance). (b) RT–qPCR analysis for the same markers as in (a) on hepatocytes overexpressing Snail (Snail), or a control vector (Ctr), and both HOTAIR (siHotair) or GFP (siCtr) silenced. RNAs were collected 72 h after infection and 48 h after transfection. The values are calculated as in (a). Statistically significant differences are reported (*P<0.05, **P<0.01) for five independent experiments. (c) Phase-contrast and immunofluorescence analysis for the indicated markers in cells as in (b) (magnification × 20). Blue DAPI staining shows the nuclei (DNA).

Figure 3

Identification of the Snail/HOTAIR/EZH2 complex. (a) RIP assays with rabbit polyclonal anti-Snail, anti-EZH2 or preimmune IgG on 24 h TGFβ-treated (TGFβ) or untreated (NT) murine hepatocyte cell extracts. RNA levels in immunoprecipitates were determined by qRT–PCR. HOTAIR lncRNA and, as controls, ribosomal L34 RNA, GAPDH pre-mRNA and SRA lncRNA were reported as percentage with respect to 1/10th of the input sample (TGFβ treated in the hotair panel or NT in the other panels) (% Input). Data are means ±s.e.m. of three independent experiments. (b) Co-immunoprecipitation of Snail and EZH2. Immunoprecipitations with rabbit polyclonal anti-Snail, anti-EZH2, preimmune IgG or no antibody (NoAb) were performed on protein extracts from murine hepatocyte cells treated with TGFβ for 24 h and silenced for HOTAIR (siHotair) or GFP (siCtr) as control. Immunoblots were performed using anti-Snail and anti-EZH2 antibodies. (c) RIP assays with rabbit polyclonal anti-Snail, anti-EZH2 or preimmune IgG on TGFβ-treated (TGFβ) or untreated (NT) human hepatoma cell extracts. RNA levels in immunoprecipitates were determined by RT–qPCR. HOTAIR lncRNA and, as controls, ribosomal L34 RNA, GAPDH pre-mRNA and SRA lncRNA were reported as percentage with respect to 1/10th of the input sample (TGFβ treated in the hotair panel or NT in the other panels) (% Input). Data are means ± s.e.m. of three independent experiments. (d) Co-immunoprecipitation of Snail and EZH2. Immunoprecipitations with rabbit polyclonal anti-Snail, anti-EZH2 or preimmune IgG were performed on protein extracts from human hepatoma cells treated with TGFβ for 24 h and silenced for HOTAIR (siHotair) or GFP (siCtr) as control. Immunoblots were performed using anti-Snail and anti-EZH2 antibodies.

Figure 4

Identification of the Snail/HOTAIR/EZH2 complex within the chromatin. (a) RNA pull-down for HOTAIR in the ChIRP analysis. Nuclei were prepared from cells overexpressing HOTAIR (HOTAIR), Snail (Snail) or both (HOTAIR/Snail) and, as control, transfected with the empty vectors (Ctr). Biotinylated complementary DNA probes effectively retrieved HOTAIR RNA, as compared with L34 and GAPDH. Note that in the double Snail/HOTAIR transfection the amount of each plasmid was halved. All the experiments have been performed in triplicate, after UV crosslinking (+UV, in denaturing condition) or not (−UV, in non-denaturing condition). As a negative control, specific pre-ribosomal 45S probes have been used in the pull-down assays, not retrieving HOTAIR in none of the tested experimental conditions. Data were reported as percentage with respect to the Input sample (% RNA retrieved). Means±s.e.m. are shown. (b) Western blot analysis for Snail (left panels) and EZH2 (right panels) of the chromatin fraction bound to HOTAIR, showing the direct interaction between both these proteins and HOTAIR. Proteins were obtained from total cell lysates (TOT) or from chromatin pulled down with probes recognizing HOTAIR (Hotair) or pre-ribosomal RNA 45S (45S). Cells were overexpressing HOTAIR (HOTAIR), Snail (Snail), both (HOTAIR/Snail) or the empty vectors (Ctr), as in (a). All the experiments have been performed in triplicate after UV crosslinking (+UV in denaturing condition) or not (−UV, in both denaturing and non-denaturing conditions). (c) ChIRP–qPCR analysis of the DNA in the chromatin fraction bound to HOTAIR. Crosslinked samples were as in (a). Data show the enrichment of HOTAIR on the Snail consensus binding sites on the murine promoters of HNF4α, E-cadherin and HNF1α only in the presence of Snail. Timm and Snail promoters were used as negative controls. Values derived from three independent experiments are expressed as the percentage of the Input chromatin (% Input) and reported as means ± s.e.m.

Figure 5

Snail binding sites chromatin trimethylation requires HOTAIR. (a) qPCR analysis of ChIP assays with anti-Snail antibody (IP Snail) and, as control, normal rabbit IgG (IgG) on chromatin from murine hepatocyte cells silenced for HOTAIR (siHotair) or GFP (siCtr), as control, and treated (+TGFβ) or not (−TGFβ) with TGFβ for 24 h. Data show the direct recruitment of endogenous Snail on the correspondent consensus binding sites on the murine promoters of HNF4α, E-cadherin and HNF1α. Timm promoter was used as a negative control. Values derived from five independent experiments are reported as means ±s.e.m. and expressed as percentage of the Input chromatin (% Input). Statistically significant differences are reported (*P<0.05, **P<0.01, ***P<0.001). (b) qPCR analysis of ChIP assays with anti-H3K27me3 antibody (IPH3K27me3) and, as controls, normal rabbit IgG (IgG) on chromatin from murine hepatocyte cells silenced for HOTAIR (siHotair) or GFP (siCtr), as control, and treated (+TGFβ) or not (−TGFβ) with TGFβ for 24 h. Data show the enrichment of H3K27 trimethylation on the Snail consensus binding sites on the murine promoters of HNF4α, E-cadherin and HNF1α, as above. Timm promoter was used as a negative control. Values derived from five independent experiments are reported as means ± s.e.m. and expressed as the percentage of the Input chromatin (% Input). Statistically significant differences are reported (*P<0.05, **P<0.01).

Figure 6

EZH2 is recruited by Snail to its target genes by means of HOTAIR. (a) qPCR analysis of ChIP assays with an anti-EZH2 antibody (IP EZH2) and, as controls, normal rabbit IgG (IgG) on chromatin from murine hepatocyte cells silenced for HOTAIR (siHotair) or GFP (siCtr), as control, and treated (+TGFβ) or not (−TGFβ) with TGFβ for 24 h. Data show the recruitment of EZH2 on the Snail consensus binding sites on the murine promoters of HNF4α, E-cadherin and HNF1α and, as control, its displacement from the HNF4α binding site on Snail promoter. Timm promoter was used as unresponsive control sequence. Values derived from five independent experiments are reported as means ± s.e.m. and expressed as the percentage of the Input chromatin (% Input). Statistically significant differences are reported (*P<0.05; **P<0.01; ***P<0.001). (b) qPCR analysis of ReChIP assays of samples TGFβ treated for 24 h and silenced for HOTAIR (siHotair) or GFP (siCtr), as control. Values, derived from three independent experiments, are reported as means ±s.e.m. and expressed as the percentage of the Input chromatin (% Input).

Figure 7

Scheme of the proposed repression switch occurring in hepatocyte undergoing EMT. (a) In mesenchymal cells Snail represses E-cadherin and HNFs through the enrollment of the HOTAIR/polycomb complex. (b) Summary of transcriptional modulation occurring during EMT.