Figure 4: Suppression of neuronal differentiation-associated genes by the TUG1-PRC2 complex.

(a) RNA pull-down analysis with anti-BrU antibody. Nuclear extracts from GSCs were incubated with TUG1 RNA labelled with BrU (BrU+) or without (BrU-). TUG1-RNA-binding proteins were analysed by western blotting. Input extract is used as control. (b) Nuclear extracts from GSCs treated with either si-NC or si-TUG1 were immunoprecipitated with anti-YY1 antibody. The immunoprecipitated fractions and lysate aliquots (Input) were subjected to western blotting. A part of the nuclear extract from si-NC-treated cells was also treated with RNase, immunoprecipitated with anti-YY1 antibody, and analysed by western blotting (RNase, left). Relative intensity values of EZH2 normalized to Input are indicated in (c). (d) Venn diagram shows relationship between TUG1 target genes (630 genes) and 1,714 genes that contain a YY1 motif around their TSS. Among 630 TUG1 target genes, 525 genes contain a YY1 motif. (e) Heatmap indicates expression changes of 525 TUG1 target genes containing a YY1 motif (shown in d) upon inhibition of TUG1 (si-TUG1 #1 and #2). Colour corresponds to expression level as indicated in the log₂-transformed scale bar below the matrix. Red and blue reflect high and low levels, respectively. (f) Averaged expression value of 525 genes shown in (e). Error bars indicate s.e.m. *P<0.001, Student’s t-test. (g) Enrichment of K27me3 in the BDNF, NGF and NTF3 promoter regions (left), and their mRNA expression levels (right) in GSCs treated with either si-NC, si-TUG1 or si-YY1. K27me3 enrichment is expressed as a percentage of input DNA (left). Relative expression level to si-NC is shown (right). Error bars indicate s.d. (n=3). *P<0.01. Kruskal–Wallis analysis. (h) Protein expression levels of BDNF and NGF in GSCs treated with either si-NC, si-TUG1 or si-YY1. (i) Expression changes of YY1 in GSCs treated with si-YY1. Values are indicated relative to abundance in si-NC-treated cells. Error bars indicate s.d. (n=3)*P<0.01, Student’s t-test. (j) ChIRP analysis for the BDNF, NGF and NTF3 promoter regions. Probes targeting EGFP-ORF were used as a negative control. GAPDH served as a non-TUG1 target gene control. Enrichment of TUG1 expressed as a percentage of input DNA. Error bars indicate s.d. (n=3) *P<0.01, Kruskal–Wallis analysis.