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. 2015 Aug;185(8):2206–2218. doi: 10.1016/j.ajpath.2015.04.023

Figure 5.

Figure 5

ChIP analysis of CLDN7 promoter binding. A: Representation of the CLDN7 promoter. Predicted transcription factor binding (TFB) sites are marked with the symbol of the TF above the promoter, their respective distance from the start ATG is indicated below their symbol. PCR products detecting specific TF/DNA interactions in chromatin immunoprecipitation (ChIP) samples are indicated under the promoter and numbered 1 to 3 and C1 to C2. B: ChIP was performed using HNF-4α, PU.1, and Oct2.1 antibodies, and PCR was performed using primers flanking the predicted binding sites of Oct2.1, HNF-4α, and PU.1. A representative gel image of two independent experiments is shown. C: ChIP was performed using an antibody to HNF-4α and immunoprecipitated DNA was analyzed by real-time PCR. Data are expressed relative to DNA isolated from the same amount of input chromatin. Note the enrichment of HNF-4α at primer sets spanning the predicted binding motifs of HNF-4α compared with parallel samples precipitated using a nonspecific IgG. Three independent experiments were performed in triplicate. Data shown are the average ± SD from a representative experiment. D: HNF-4α is enriched at the CLDN7 promoter in differentiated compared with nondifferentiated Caco-2 cells. Data shown are the means ± SD of one experiment assayed in triplicate. E: HNF-4α shows a gradient similar to claudin-7, with the highest expression in differentiated cells in differentiating intestinal epithelial cells (IECs). Western blots of Caco-2 cell lysates are shown. Analogous to model IECs, gradients were observed in the expression of claudin-7 and HNF-4α in cryosections of human colonic mucosa by immunofluorescence labeling and laser confocal microscopy. Claudin-7 and HNF-4α are shown in green in F and H, and G and I, respectively. Notice the higher expression of both proteins on the surface (F and G) compared with the crypt base (H and I). Scale bar = 50 μm (F–I). F-actin was labeled with Alexa phalloidin in the HNF-4α images. IP, immunoprecipitation; UTR, untranslated region.