Figure 3

Smad3 binding differs widely across the progression series. (A) Petal plot showing the overlap in Smad3 target genes between the different cell lines. The genes involved are given in Supporting Information Table S1 in Additional file 4. (B) Representative genes with distinct Smad3 occupancy patterns as confirmed by ChIP-QPCR. Closed circles indicate TGF-β-induced Smad3 occupancy. (C) DNA methylation status at promoter regions of each target gene in (B) as determined by MeDIP-QPCR. Relative enrichment in the bound (MeDIP) vs. unbound fractions is shown. PPIA and MyoD were controls for unmethylated and highly methylated DNAs respectively. (D) QPCR quantitation of target enrichment following ChIP using anti-H3AcK9/14 to identify active chromatin. Enrichment at the SBR was calculated relative to input DNA. PPIA and MyoD were controls for active and inactive promoters respectively. Active chromatin has an enrichment value >1.00 (indicated by threshold line). ChIP, chromatin immunoprecipitation; H3AcK9/14, histone H3 acetylated on lysine 9 or 14; MeDIP, methylated DNA immunoprecipitation; QPCR, quantitative polymerase chain reaction; SBR, Smad binding region; TGF-β, transforming growth factor beta.