Figure 4.

AmeloD binds to E-cadherin proximal promoter and inhibits E-cadherin expression. (A) ChIP-qPCR results showed that AmeloD binds to the E-cadherin proximal promoter. Nuclear extracts were prepared from CLDE cells transfected by adeno-AmeloD for 72 h. Antibodies for AmeloD and IgG were used for ChIP assay. Primer set P1-P2 was designed to amplify the proximal promoter region (−69 bp to TSS) of the mouse E-cadherin gene, and the primer set P3-P4 was designed to amplify 100 bp of the E-cadherin gene exon 2. (B) Left panel: ChIP-qPCR results showed that binding of AmeloD to the E-cadherin promoter increased the repressive marker H3K27me3 enrichment. The H3k27me3 enrichment level was relative to the percentage of input. Nuclear extracts prepared from CLDE cells were transfected by adeno-GFP and adeno-AmeloD expression vectors for 72 h. Antibodies for H3K27me3 and IgG were used for immunoprecipitation. Right panel: ChIP-qPCR results showed that binding of AmeloD to the E-cadherin promoter increased the PCR2 complex component Ezh2 enrichment. The Ezh2 enrichment level was relative to the percentage of input. Nuclear extracts prepared from CLDE cells were transfected by adeno-GFP and adeno-AmeloD expression vectors for 72 h. Antibodies for Ezh2 and IgG were used for immunoprecipitation. (C) The upper panel shows that the consensus E-box element (E1 and E2) appears at the E-cadherin proximal promoter among different species; the lower panel shows the diagram of the wild-type (WT) and mutated E-cadherin luciferase reporter. (D) Luciferase assay showed that AmeloD inhibits the WT E-cadherin luciferase reporter activity but not the mutated E-cadherin luciferase reporter. Transcription factor snail2 served as a positive control, and an empty construct served as a negative control. Data are shown as mean ± SD (n = 3). Statistical significance is shown by a t test. *P < 0.05. ChIP, chromatin immunoprecipitation; qPCR, quantitative polymerase chain reaction.