Using In Vivo Footprinting To Determine Altered Transcription Factor Binding After Phthalate Treatment
Kuhl, A; Ross, S; Gaido, K
| HERO ID | 1323106 |
|---|---|
| In Press | No |
| Year | 2006 |
| Title | Using In Vivo Footprinting To Determine Altered Transcription Factor Binding After Phthalate Treatment |
| Authors | Kuhl, A; Ross, S; Gaido, K |
| Journal | Toxicological Sciences |
| Volume | 90 |
| Issue | 1-S |
| Abstract | Phthalate esters, including di(n-butyl) phthalate (DBP), are endocrine active compounds that can adversely affect male reproductive development at relatively high doses in the rat after in utero exposure. DBP effects include cryptorchidism, hypospadias, and reduced semen quality, which can be attributable in part to reduced testosterone biosynthesis in the fetal testis. In utero phthalate exposure of fetal testis on gestation days 12 ? 19 results in a reduction of key steroidogenic enzymes mRNA and protein levels including StAR, SR-B1, P450scc, and CYP17. However, the mechanism of this reduction is unknown. We hypothesize that phthalate exposure results in altered transcription factor binding in the promoter regions of affected genes. To examine this hypothesis, an in vivo footprinting method was employed to examine differences in transcription factor binding in steroidogenic gene promoters after phthalate treatment. In vivo footprinting employs ligation-mediated PCR to amplify single-sided DNA created by strand breaks after treatment with DNase I in the promoter sequence of the gene. Regions bound by transcription factors are protected from the DNase I and therefore not amplified. This method was adapted for use with an automated DNA sequencer to allow greater sensitivity and quantification. Results indicate that phthalate treatment induces a reduction in transcription factor binding between -35 and -70 bp upstream of the transcriptional start site in the proximal promoter region of StAR. Sequence analysis of this region reveals two highly consensus binding sites for GATA binding proteins as well as a half-site for Steroidogenic Factor-1 (SF-1). The disruption of activity of transcription factors known to be highly involved in activation of steroidogenic genes suggests a possible mechanism through which phthalates reduce testosterone production. |
| Is Certified Translation | No |
| Dupe Override | 1323106 |
| Is Public | Yes |
| Language Text | English |