Coexamination of Site-Specific Transcription Factor Binding and Promoter Activity in Living Cells

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Abstract
Previously, we have used a chromatin cross-linking and immunoprecipitation protocol for the analysis of Myc and USF binding to the cad promoter. The adaptation of this technique for the study of mammalian transcription factors was a big step forward in the analysis of transcription factor family member specificity, allowing for the first time a definitive knowledge of which factor binds to a promoter region under normal physiological conditions. However, due to limitations of the assay, our previous studies could not definitively prove that both Myc and USF bound to the exact same site on the cad promoter, nor could we directly correlate loss of in vivo binding of a particular factor with loss of transcriptional activity. Therefore, we have further modified the chromatin immunoprecipitation protocol to alleviate these problems. We have now shown that it is possible to coexamine growth-regulated transcriptional activity and promoter occupancy by using stably integrated promoter constructs. We show that both Myc and USF bind to the exact same E box on the cad promoter, suggesting that competition between these two factors for a single site occurs in living cells. We also find that cad promoter constructs that retain USF binding but lose Myc binding in vivo no longer display an increase in transcriptional activity in mid- to late G1 phase of the cell cycle. Finally, we propose that cell cycle-regulated transcriptional activation of the cadpromoter may be a stochastic, rather than a predetermined, process.