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. 2007 Oct 8;27(23):8164–8177. doi: 10.1128/MCB.00555-07

FIG. 7.

FIG. 7.

Nonresponsive Wnt target genes remain refractory to Wnt induction upon inhibition of histone deacetylases or DNA methyltransferases. (A) TSA increases the basal expression level but does not restore the Wnt inducibility of the T/Bra gene. C17.2 cells were treated for 24 h with 0.5 μM TSA or 5 μM Aza in combination with 50 ng/ml Wnt3a purified from conditioned medium (lanes 1 to 6) as indicated. Total RNA was isolated, and the activation of the Wnt target genes Axin2, Cdx1, and T/Bra was analyzed by RT-PCR. Amplification of GAPDH was used to control sample integrity and loading. Control reactions were done as described for Fig. 2A. (B) DNA methylation analysis of the T/Bra promoter in C17.2 cells upon TSA and Aza treatment. Bisulfite-treated DNA was amplified by PCR with primers indicated in Fig. 1 and analyzed as described before (Fig. 5). (C) Treatment with TSA increases levels of acetylated histone H3. Immunoprecipitations with anti-acetylated H3 (α-acH3) antibodies were performed with nuclear lysates from C17.2 cells treated for 24 h with 0.5 μM TSA, 5 μM Aza, or DMSO as the solvent control. A fraction of the nuclear (nucl.) lysates (2.5%) and the immunoprecipitates were analyzed by immunoblotting with anti-acetylated H3 antibodies. Coomassie brilliant blue staining of nuclear lysates separated by SDS-PAGE was used to confirm equal loading. (D) Inhibition of histone deacetylases does not alter the epigenetic states and TCF promoter occupancy of Wnt target genes. ChIP analysis for TCF4, acetylated histone H3, and methylated histone H3K27 in TSA-treated C17.2 cells. Formaldehyde-fixed chromatin was prepared from C17.2 cells after treatment with 1 μM TSA or DMSO as the solvent control for 24 h. Chromatin was precipitated using 2 μg of a TCF4-specific antibody, antibodies to acetylated histone H3 (2 μg) or trimethylated histone H3K27 (α-H3K27me3) (4 μg), or equivalent amounts of the corresponding isotype controls. Precipitated DNA was analyzed by quantitative real-time PCR using primer sets within the promoter regions of the Axin2, Cdx1, and T/Bra genes. Nucleotide positions of the amplicons relative to the transcriptional start site are indicated (Fig. 1). Bars represent relative enrichment compared to that of the nonspecific isotype controls (relative enrichment of 1). Data given are the average and SEM from at least three experiments.