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.