Detection of interaction of binding affinity of aromatic hydrocarbon receptor to the specific DNA by exonuclease protection mediated PCR assay

Author(s):  
Sun Xi ◽  
Xu Shunqing
1996 ◽  
Vol 16 (1) ◽  
pp. 430-436 ◽  
Author(s):  
H P Ko ◽  
S T Okino ◽  
Q Ma ◽  
J P Whitlock

We have analyzed the dioxin-inducible transcriptional control mechanism for the mouse CYP1A1 gene in its native chromosomal context. Our genetic and biochemical studies indicate that a C-terminal segment of the aromatic hydrocarbon receptor (AhR) contains latent transactivation capability and communicates the induction signal from enhancer to promoter. Thus, transactivation and enhancer-promoter communication may be congruent functions of AhR. Both functions require heterodimerization between AhR and the AhR nuclear translocator (Arnt). Our findings also indicate that heterodimerization activates AhR's latent transactivation function and silences that of Arnt. Furthermore, removal of Arnt's transactivation domain does not affect dioxin-induced CYP1A1 transcription in vivo. In addition, our studies demonstrate that dioxin-induced changes in chromatin structure occur by different mechanisms at the CYP1A1 enhancer and promoter and that events at an enhancer can be experimentally dissociated from events at the cognate promoter during mechanistic analyses of mammalian transcription in vivo.


2002 ◽  
Vol 33 (9) ◽  
pp. 1268-1278 ◽  
Author(s):  
Albert P Senft ◽  
Timothy P Dalton ◽  
Daniel W Nebert ◽  
Mary Beth Genter ◽  
Alvaro Puga ◽  
...  

10.1038/ng575 ◽  
2001 ◽  
Vol 28 (4) ◽  
pp. 355-360 ◽  
Author(s):  
Tiina Matikainen ◽  
Gloria I. Perez ◽  
Andrea Jurisicova ◽  
James K. Pru ◽  
Jennifer J. Schlezinger ◽  
...  

2010 ◽  
Vol 140 (9) ◽  
pp. 1607-1614 ◽  
Author(s):  
Andreas J. Papoutsis ◽  
Sarah D. Lamore ◽  
Georg T. Wondrak ◽  
Ornella I. Selmin ◽  
Donato F. Romagnolo

2003 ◽  
Vol 81 (1) ◽  
pp. 59-77 ◽  
Author(s):  
David S Riddick ◽  
Chunja Lee ◽  
Anahita Bhathena ◽  
Yoav E Timsit

Most responses to aromatic hydrocarbons such as 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin are mediated by the aromatic hydrocarbon receptor (AHR). The AHR regulates induction of drug-metabolizing enzymes such as cytochrome P450 1A1. However, the expression of several genes of biological significance is decreased by these chemicals. We are examining the mechanisms by which aromatic hydrocarbons suppress constitutive hepatic cytochromes P450, especially the male-specific rat liver cytochrome P450 2C11 (CYP2C11), which is regulated by pulsatile growth hormone (GH) secretion. Aromatic hydrocarbons suppress CYP2C11 via a transcriptional mechanism both in vivo and in cultured hepatocytes, and the AHR appears to be involved; however, studies of protein–DNA interactions and reporter genes driven by the CYP2C11 5'-flanking region have not provided a definitive mechanism for this response. MC attenuates the ability of GH to stimulate hepatic CYP2C11 expression in hypophysectomized (hypx) male rats, and this prompted studies of effects of aromatic hydrocarbons on hepatic GH signaling pathways as a novel aspect of endocrine disruption. Our studies with hypx rats also suggest that the hepatic AHR protein is regulated by a pituitary factor(s). The goal of these molecular mechanistic studies is to improve our understanding of how environmental contaminants modulate the expression of genes coding for xenobiotic- and hormone-metabolizing enzymes.Key words: aromatic hydrocarbons, cytochrome P450, aromatic hydrocarbon receptor, growth hormone, transcriptional regulation.


1995 ◽  
Vol 15 (7) ◽  
pp. 3714-3721 ◽  
Author(s):  
S T Okino ◽  
J P Whitlock

In mouse hepatoma cells, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) induces Cyp1A1 gene transcription, a process that requires two basic helix-loop-helix regulatory proteins, the aromatic hydrocarbon receptor (AhR) and the aromatic hydrocarbon receptor nuclear translocator (Arnt). We have used a ligation-mediated PCR technique to analyze dioxin-induced changes in protein-DNA interactions and chromatin structure of the Cyp1A1 enhancer-promoter in its native chromosomal setting. Dioxin-induced binding of the AhR/Arnt heteromer to enhancer chromatin is associated with a localized (about 200 bp) alteration in chromatin structure that is manifested by increased accessibility of the DNA; these changes probably reflect direct disruption of a nucleosome by AhR/Arnt. Dioxin induces analogous AhR/Arnt-dependent changes in chromatin structure and accessibility at the Cyp1A1 promoter. However, the changes at the promoter must occur by a different, more indirect mechanism, because they are induced from a distance and do not reflect a local effect of AhR/Arnt binding. Dose-response experiments indicate that the changes in chromatin structure at the enhancer and promoter are graded and mirror the graded induction of Cyp1A1 transcription by dioxin. We discuss these results in terms of a TCDD-induced shift in an equilibrium between nucleosomal and nonnucleosomal chromatin configurations.


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