Fig. 3
ZEB1-AS1 bound to and recruited MLL1 to maintain H3K4me3 modification in ZEB1 promoter region. A and B, ChIP showed H3K4me3 modification in ZEB1 promoter which was mediated by ZEB1-AS1. PCR using chromatin pulled down by H3K4me3 antibody as template yielded the ZEB1-pro fragment (−1 to −103) of the ZEB1 promoter. ZEB1-AS1 knockdown (ZEBAS1-KD1 and ZEBAS1-KD2) in both DU145 and PC3 cells similarly induced decreased ZEB1 promoter fragment amplified from the precipitates (a). In addition, adenovirus-mediated ZEB1-AS1 re-expression (AD-ZEBAS1) rescued this effect (b). c and d, MLL1 mediated the transcription of ZEB1 by combing with its promoter. RT-PCR showed that MLL1 interfering (si-MLL1) inhibited ZEB1 mRNA level in both DU145 and PC3 (c). The corresponding ChIP using chromatin pulled down by MLL1 antibody as template showed similar ZEB1-pro attenuation (d) as in a. e, ChIP showed that H3K4me3 modification in ZEB1 promoter region was mediated by MLL1. MLL1 interfering (si-MLL1) in both DU145 and PC3 cells similarly induced decreased ZEB1 promoter fragment amplified from the precipitates. f, RIP showed combination of ZEB1-AS1 and MLL1. RT-PCR using RNAs pulled down by MLL1 antibody yielded ZEB1-AS1 fragment. Significant decrease of ZEB1-AS1 level was detected in MLL1 interfering cells (si-MLL1, ZEBAS1-KD2 as negative control)