Dioxin- and Ah receptor-dependent protein binding to xenobiotic responsive elements and G-rich DNA studied by in vivo footprinting.

A.J Watson,O Hankinson

doi:10.1016/s0021-9258(19)50509-9

Abstract

DNA-protein interactions before and after transcriptional activation of the carcinogen- and dioxin-inducible enhancer of the murine CYP1A1 gene were detected in vivo by treatment with dimethyl sulfate followed by ligation-mediated, polymerase chain reaction-aided genomic sequencing. Following 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment of mouse Hepa-1 hepatoma cells, evidence of protein binding was detected at the sequence 5' CACGCNA/T 3' within two previously defined xenobiotic response elements (XREs). The observed XRE footprint was similar to that previously identified by in vitro methylation protection footprints and attributed to the binding protein for 2,3,7,8-tetrachlorodibenzo-p-dioxin the Ah receptor. No XRE footprinting was observed in Hepa-1 mutant cells possessing a defective Ah receptor. Unexpectedly, evidence of protein binding was also detected at a G-rich DNA sequence immediately adjacent to one of the XREs. Footprinting of the G-rich sequence element, like that of XRE1 and XRE2, was dependent on the presence of a functional Ah receptor. The Ah receptor is therefore able to bind to its own DNA target sites in vivo and is also required for the binding of a second factor to the G-rich element.

Highlights

DNA transfection of mouse hepatoma Hepa-1 cells with inducible enhancer of the murineCYPlAl gene were reporter gene constructs consisting of truncated versions of detected in vivo by treatmentwithdimethylsulfate rodent CYPlAl enhancers fused to the bacterial chloramfollowed by ligation-mediated, polymerase chain re- phenicol acetyltransferase gene have revealed the existence action-aided genomic sequencing
P450IA1 expression is inducible by polycyclic aromatic hydrocarbons andstructurally related compounds suchas 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD),’ and is regulated at the transcriptional level by a high affinity cytosolic receptor protein for inducing compounds, the aryl hydrocararecontacted by protein [10, 14, 15]
On the basis of these results, we propose that the CYPlAl enhancer possesses a modular structure consistreaction; bp, base pairs; GRB, G-rich box

Summary

EXPERIMENTAL PROCEDURES

Chmicak-Dimethyl sulfate and piperidine were obtained from Aldrich. TCDD was obtained from the National Cancer Institute Chemical Carcinogen Repository. The cell suspensions were pelleted quickly in a microfuge a t 4 "C and resuspended in 0.5ml of cold growth medium containing 0.5% of freshly added dimethyl sulfate. DNA methylated in uiuo by either method was purified as described [23]. Preparation of in vitro methylated Hepa-1 genomic DNA was by standard methods [29]. Labeled reaction products were purified, fractionated on standard 6%sequencing gels, and visualized by autoradiography. Both DNA strands were footprinted using three specific primers in addition to theuniversal linker primers. The labeling (primer 3) reactions were carried out at 70 "C (annealing) and 80 "C (extension). The numbering used throughout this paper reflects our besteffort to resolve the different published sequences [6, 30] with the sequence ladders we obtained from pHAVcat plasmid [3] and Hepa-1 cellular DNA

RESULTS AND DISCUSSION

HMM v

GHHM M

In VivoFootprinting of the Mouse CYPlAl Enhancer