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Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A

h continuous SYBR Green I fluorescence monitoring (Morrison et al. 1998) offers the level of sensitivity and quantitation necessary to study transcription factor-DNA interactions in the context of the mammalian genome.

The mammalian transcription factor Pdx1 (also known as IPF1) is expressed almost exclusively in β-cells in the mature mouse pancreas. β-cells express a unique set of genes, including insulin, glucose transporter type 2 (Glut2), islet amyloid polypeptide (IAPP), and glucokinase (GK). Several functional studies have implicated a direct role for Pdx1 in the specific activation of these genes (Ahlgren et al. 1998). However, none of these functional studies have established whether Pdx1 is involved in a transcriptional complex that directly controls the expression of these genes. Since chromatin structure within a nuclear environment can influence both the binding of transcription factors to specific DNA targets and the resultant transcription rate, we cannot know from just functional and in vitro binding studies which genes Pdx1 natively regulates. To determine which promoters Pdx1 might directly regulate in vivo, we performed ChIP assays using Pdx1 antiserum and chromatin from Pdx1-transfected NIH 3T3 cells (NIH 3T3/Pdx1) and from empty vector-transfected NIH 3T3 cells (NIH 3T3/EV). Immunoprecipitated DNA fragments were detected by quantitative real-time PCR using SYBR Green I and the Bio-Rad iCycler iQ system.


Method
Chromatin immunoprecipitation assays using NIH 3T3/Pdx1 and NIH 3T3/EV cells were performed as previously described (Kuo and Allis 1999). Primers for the PCR reactions were designed using the MacVector software package (
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