( iserum reaches the threshold...)Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A

iserum reaches the threshold approximately three cycles before control immunoprecipitations (which included NIH 3T3/Pdx1 chromatin immunoprecipitated with normal rabbit serum and NIH 3T3/EV chromatin immunoprecipitated with Pdx1 antiserum or normal rabbit serum). This finding (ΔΔC_T = 3) implies an approximately 8-fold (i.e., 2³) enrichment of insulin promoter in the NIH 3T3/Pdx1 immunoprecipitation compared to NIH 3T3/EV immunoprecipitations. Each value was normalized to the total input DNA (i.e., total genomic DNA), to correct for variations in input quantity of DNA prior to immunoprecipitation (as detailed in Method). The results shown in Figure 4 summarize the real-time PCR results for each of the promoters considered in this study.

Figure 4 demonstrates that in NIH 3T3/Pdx1 cells, Pdx1 shows 825-fold greater association with the insulin, IAPP, glucokinase, glucagon, and Pax4 promoters than in controls. While an approximately 7-fold greater association of Pdx1 with the Pdx1 promoter was observed, this was not statistically significant (P = 0.07). Pdx1 displays no statistically significant association with the Glut2 and albumin promoters, notwithstanding that these promoters contain potential high-affinity binding sites for Pdx1 based on in vitro binding assays. Moreover, the Glut2 promoter is believed to be a direct binding target for Pdx1 based on functional assays in cell lines (Waeber et al. 1996). Thus, our studies suggest that the DNA binding properties of Pdx1 in vitro do not necessarily predict its binding properties on chromatin templates in vivo. As expected in these studies, NIH 3T3/EV cells show no significant coimmunoprecipitation of any of the
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