Figure 1.
Identification of a functional AR-regulated AS noncoding RNA in the CTBP1 locus. (A) CAGE TCs significantly regulated by androgen were extracted (Takayama et al, 2011). Among 125 Refseq genes with androgen-regulated AS TCs, 39 genes also had androgen-regulated TCs in the sense direction. Of these sense-AS pairs, 11 included the genes adjacent to ChIP-chip-determined ARBSs (Takayama et al, 2011). The heat map represents CAGE tag counts. (B) Mapping of ChIP-chip and CAGE data in the CTBP1 locus. (C) Chromatin immunoprecipitation (ChIP) analysis of AR. LNCaP or VCaP cells were treated with 1 nM R1881 or vehicle. Cell lysates were immunoprecipitated with rabbit IgG or anti-AR antibody. Bar: s.d. (D, E) CTBP1 ARBS mediates transcriptional activity. A 2-kb fragment that included the CTBP1 ARBS was cloned into the pGL3 luciferase vector (D). Two noncanonical AREs were mutated in the Mut #1 and Mut #2 vectors. A luciferase assay was performed in LNCaP cells with the indicated treatments for 24 h (E). Bar: s.d. (F) Reciprocal CTBP1 and CTBP1-AS expression at the indicated time points after androgen treatment (n=3). (G) Protein level regulation of CTBP1 in LNCaP cells treated with R1881 or vehicle. (H) Schematic view of the CTBP1-AS transcripts obtained by our RACE PCR analysis. The location of the RNA probe is indicated. (I) Northern blot analysis of CTBP1-AS or β-actin by using total RNA from cells treated with androgen. (J) qRT–PCR analysis for transcriptional regulation of CTBP1 in VCaP cells after androgen treatment. Bar: s.d. (K, L) Total RNAs from nuclear and cytoplasmic cell fractions were used for qRT–PCR (n=2) (K) and northern blot (L) analysis. TMPRSS2 is an androgen-induced protein-coding gene used as a positive control. (M) RNA FISH of CTBP1-AS in LNCaP cells. Bar=10 μm. (N) siRNA-mediated knockdown of CTBP1-AS (N=3). (O) siCTBP1-AS-dependent changes in androgen-stimulated expression of CTBP1 protein in LNCaP cells.
Source data for this figure is available on the online supplementary information page.
