Repression of ZBP1 expression in metastatic cells results from promoter
methylation, which prevents the binding of the promoter to β-catenin. (A)
In vitro methylation of the ZBP1 promoter leads to repression of its
transcriptional activity. Three luciferase constructs were in vitro methylated
by SssI methyltransferase. Left: completion of in vitro methylation was
evaluated by restriction digestion of methylated (lane 1) or unmethylated
(lane 2) constructs with EagI, which recognizes unmethylated CGGCCG
sequences. Right: after transfecting the reporters into mTC cells, luciferase
activity was measured and normalized. Relative luciferase activity is
represented as a percentage of the unmethylated pSV-40 construct. Values are
the average of two independent assays ± s.e.m. (B) Western blot
analysis of ZBP1 and β-actin protein expression in MTC and MTLn3 cells
treated or non-treated with 5-Aza-dC. The arrows indicate the positions of the
detected proteins. (C) Analyses of ZBP1 and β-actin mRNA
expression in the same cell cultures as shown in B. The arrows indicate the
positions of the ZBP1 and β-actin transcripts. (D) MTC cells and
MTLn3 cells incubated with or without 5-Aza-dC were subjected to chIP using
antibodies against acetylated histone and β-catenin. Total nuclear
lysates were used as an input control to purified chromatin DNA. Two primer
pairs, one amplifying a region of the ZBP1 promoter containing a
putative CTTTG-TC binding site for β-catenin (upper) and the other
amplifying a region 2-kb upstream of the ZBP1 transcription site
(lower), were used in the experiments. Lanes 1, 4 and 7: inputs of MTC cells,
MTLn3 cells and MTLn3 cells treated with 5-Aza-dC. Lanes 2, 5 and 8:
precipitates with antibodies against acetylated histone. Lanes 3, 6 and 9:
precipitates with antibodies against β-catenin. Anti-β-catenin
antibody specifically pulled down the ZBP1 promoter in MTC and
5-Aza-dC-treated MTLn3 cells, but not in parental MTLn3 cells because of the
promoter methylation.