Using a comparative in vivo DNase I footprinting technique to analyze changes in protein-DNA interactions following phthalate exposure

Exposure to environmental chemicals often induces changes in gene expression leading to a variety of developmental and physiological problems. Understanding the underlying mechanism of these changes will aid in assessing human risk to these chemicals. Traditional methods for analyzing protein–DNA interactions include in vivo footprinting and chromatin immunoprecipitation (ChIP). However, ChIP does not provide binding location, and conventional footprinting is too subjective and time consuming for comparing protein binding in toxicological studies. Here, in vivo DNase I footprinting is adapted for use with the automated DNA sequencer to provide a semiquantitative map of changes in DNA–protein interactions in the promoter of steroidogenic acute regulatory (StAR) protein. StAR is the rate‐limiting step in testosterone biosynthesis and is downregulated following in utero di‐butyl phthalate (DBP) treatment in rats through an unknown mechanism. In vivo footprinting identified three regions of altered DNase digestibility following DBP treatment, and EMSA identified the corresponding transcription factors as SF‐1, c/ebp β, and GATA4. ChIP assays confirmed changes in protein‐binding activity of SF‐1 and c/ebp β, but only c/ebp β gesponds to only DBP. This suggests that c/ebp β ginding is involved in DBP‐induced transcriptional changes. By tailoring in vivo footprinting for toxicological studies, it can provide a detailed and accurate map of protein–DNA interactions and is an excellent first step in determining the changes in the structure of transcriptional machinery following an exogenous chemical treatment. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:312–322, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20192