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 beta, and GATA4. ChIP assays confirmed changes in protein-binding activity of SF-1 and c/ebp beta, but only c/ebp beta gesponds to only DBP. This suggests that c/ebp beta 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.
Kuhl
Adam J
AJ
The Hamner Institutes for Health Sciences (Formerly CIIT Centers for Health Research), 6 Davis Drive, PO Box 12137, Research Triangle Park, NC 27709, USA. akuhl@ciit.org
Ross
Susan M
SM
Gaido
Kevin W
KW
D000818:Animals; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D003993:Dibutyl Phthalate / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D008297:Male; D010750:Phosphoproteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013737:Testis / Q000187:drug effects / Q000378:metabolism; D013739:Testosterone / Q000096:biosynthesis; D014157:Transcription Factors / Q000378:metabolism; D000098682:Steroidogenic Acute Regulatory Protein
D010750:Phosphoproteins; D011506:Proteins; D014157:Transcription Factors; D000098682:Steroidogenic Acute Regulatory Protein; D003993:Dibutyl Phthalate; D013739:Testosterone; D004247:DNA; D003850:Deoxyribonuclease I