Research Interests

Dioxin acts by altering the transcription of specific genes; the chemical serves as a prototype for environmental contaminants that elicit responses via receptor-dependent mechanisms. As a model response for studying dioxin action, we analyze the mechanism by which TCDD induces the transcription of the CYP1A1 gene. Cyp1A1 encodes cytochrome P450 1A1, a microsomal enzyme that oxygenates certain lipophilic aromatic hydrocarbons, such as the carcinogen benzo(a)pyrene, during their metabolic processing to water-soluble derivatives. We use mouse hepatoma cells as an experimental system, and our studies employ a variety of biochemical, genetic, and molecular biological techniques to analyze the mechanism of gene transcription and dioxin action. Our experiments have contributed to the elucidation of a novel transcriptional control system, which involves two regulatory bHLH/PAS proteins, a variety of protein-DNA interactions and protein-protein interactions, and alterations in chromatin structure. Our findings have generated new insights into the pathways by which cells respond to environmental stimuli by altering the expression of specific genes.

click on image for area of interest:

 

[transcriptional control] [bHLH/PAS proteins] [protein-DNA interactions] [chromatin structure]
[protein-protein interactions] [hypoxia-response genes] [new dioxin-responsive genes]


Transcriptional Control

The induction of CYP1A1 gene transcription in mouse hepatoma cells constitutes a powerful experimental system for studying the regulation of mammalian gene expression. CYP1A1 encodes the microsomal enzyme cytochrome P450 1A1, which oxygenates certain lipophilic substrates, such as the carcinogen benzo(a)pyrene, during their metabolic processing to water-soluble derivatives. The induction of CYP1A1 transcription is robust and occurs in cells in culture, which facilitates the use of gene transfer techniques for analyzing the induction mechanism. The existence of mutants, which are defective in the bHLH/PAS proteins, AhR and Arnt, permits a combined biochemical and genetic strategy in designing mechanistic experiments. The use of ligation-mediated PCR methods for studying protein-DNA interactions in intact cells and highly efficient retroviral vectors for gene transfer permit analyses of TCDD-responsive genes in their native chromatin structure and studies of AhR/Arnt function under physiological conditions in vivo. The use of two-hybrid systems leads to the discovery of additional factors which contribute to dioxin-inducible transcription via protein-protein interactions and to the identification of other signaling systems, such as that which regulates hypoxia-responsive genes. The differential display technique permits the discovery of new dioxin-responsive genes; their study generates novel insights into TCDD-inducible gene regulation and the biological effects of dioxin.

Recent Publications:

Whitlock JP Jr, Okino ST, Dong L, Ko HP, Clarke-Katzenberg R, Ma Q, Li H
Cytochromes P450 5: induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription.
FASEB J. 1996 Jun; 10(8): 809-818. Review.

Dong L, Ma Q, Whitlock JP Jr
Down-regulation of major histocompatibility complex Q1b gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
J Biol Chem. 1997 Nov 21; 272(47): 29614-29619. PDF full text reprint

Whitlock JP Jr
Induction of cytochrome P4501A1
Annu Rev Pharmacol Toxicol 1999; 39: 103-25.


bHLH/PAS Proteins

>After diffusing into the cell, TCDD binds to a cytoplasmic protein known as the aromatic hydrocarbon receptor (AhR). Structurally, the AhR is a member of the basic helix-loop-helix / Per-Arnt-Sim (bHLH/PAS) group of transcriptional regulatory proteins. The AhR has multiple functional domains, which mediate ligand binding, protein-protein interaction, DNA binding, and transactivation. The liganded receptor enters the cell nucleus, where it heterodimerizes with a second bHLH/PAS protein, known as the AhR nuclear translocator (Arnt). Like AhR, the Arnt protein has a modular organization and contains domains that mediate heterodimerization with AhR, DNA binding, and transactivation.

The AhR/Arnt heterodimer recognizes a specific DNA sequence and binds to multiple sites within a xenobiotic responsive enhancer (XRE) located upstream of the target CYP1A1 gene. These protein-DNA interactions are associated with alterations in both chromatin structure and protein binding at the CYP1A1 promoter. AhR's transactivation domain communicates the induction signal from enhancer to promoter probably via protein-protein interactions, thereby facilitating the binding of general transcription factors to the promoter.

Arnt can also interact with other bHLH/PAS proteins, thereby mediating responses to additional environmental stimuli. In particular, Arnt heterodimerizes with hypoxia-inducible factor 1 alpha (HIF1 alpha) in response to low oxygen tension, and the HIF1 alpha/Arnt heterodimer alters the transcription of hypoxia-responsive genes.

Recent Publications:

Li H, Dong L, Whitlock JP Jr
Transcriptional activation function of the mouse Ah receptor nuclear translocator.
J Biol Chem. 1994 Nov 11; 269(45): 28098-28105.

Ma Q, Dong L, Whitlock JP Jr
Transcriptional activation by the mouse Ah receptor. Interplay between multiple stimulatory and inhibitory functions.
J Biol Chem. 1995 May 26; 270(21): 12697-12703. full text

Ko HP, Okino ST, Ma Q, Whitlock JP Jr
Dioxin-induced CYP1A1 transcription in vivo: the aromatic hydrocarbon receptor mediates transactivation, enhancer-promoter communication, and changes in chromatin structure.
Mol Cell Biol. 1996 Jan; 16(1): 430-436.

Ma Q, Whitlock JP Jr
The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin.
Mol Cell Biol. 1996 May; 16(5): 2144-2150.

Dong L, Ma Q, Whitlock JP Jr
DNA binding by the heterodimeric Ah receptor. Relationship to dioxin-induced CYP1A1 transcription in vivo.
J Biol Chem. 1996 Apr 5; 271(14): 7942-7948. PDF full text reprint

Ko HP, Okino ST, Ma Q, Whitlock JP Jr
Transactivation domains facilitate promoter occupancy for the dioxin-inducible CYP1A1 gene in vivo.
Mol Cell Biol. 1997 Jul; 17(7): 3497-3507.


Protein-DNA Interactions

Two cis-acting DNA regulatory elements contribute to CYP1A1 gene transcription. A xenobiotic-responsive enhancer (XRE), centered about 1 kb upstream of the transcription start site, contains multiple binding sites for AhR/Arnt, a heterodimer that consists of two bHLH/PAS proteins. The protein-DNA interactions at the XRE occur at specific DNA motifs and are TCDD-inducible, AhR-dependent, and Arnt-dependent. The protein-DNA interactions produce a bend in the XRE in vitro and are associated with an alteration in its chromatin structure in vivo.

The CYP1A1 promoter, located immediately upstream of the transcription start site, contains binding sites for several general transcription factors. The protein-DNA interactions at the promoter are TCDD-inducible, AhR-dependent, and Arnt-dependent even though the promoter has no recognition motifs for the AhR/Arnt heterodimer. Induction of transcription involves communication between the enhancer and promoter and the generation of an accessible chromatin structure at the promoter. AhR's transactivation domain mediates these functions, probably via protein-protein interactions.

Recent Publications:

Okino ST, Whitlock JP Jr
Dioxin induces localized, graded changes in chromatin structure: implications for Cyp1A1 gene transcription.
Mol Cell Biol. 1995 Jul; 15(7): 3714-3721.

Dong L, Ma Q, Whitlock JP Jr
DNA binding by the heterodimeric Ah receptor. Relationship to dioxin-induced CYP1A1 transcription in vivo.
J Biol Chem. 1996 Apr 5; 271(14): 7942-7948. PDF full text reprint

Ko HP, Okino ST, Ma Q, Whitlock JP Jr
Dioxin-induced CYP1A1 transcription in vivo: the aromatic hydrocarbon receptor mediates transactivation, enhancer-promoter communication, and changes in chromatin structure.
Mol Cell Biol. 1996 Jan; 16(1): 430-436.

Ko HP, Okino ST, Ma Q, Whitlock JP Jr
Transactivation domains facilitate promoter occupancy for the dioxin-inducible CYP1A1 gene in vivo.
Mol Cell Biol. 1997 Jul; 17(7): 3497-3507.


Chromatin Structure

The enhancer/promoter region for the CYP1A1 gene assumes a nucleosomal configuration in uninduced cells, and a nucleosome is specifically positioned at the promoter. The nucleosomal organization accounts for the lack of CYP1A1 expression in uninduced cells, because nucleosomes repress transcription. Following exposure of cells to TCDD, the binding of AhR/Arnt heterodimers to the enhancer is associated with a change in chromatin structure, and enhancer DNA becomes more accessible to DNA-binding proteins. During induction, the CYP1A1 promoter undergoes loss of the positioned nucleosome and becomes more accessible to promoter-binding proteins. AhR's transactivation domain facilitates the binding of transactivation factors at the promoter's TATA box and NF1 site, perhaps by a recruitment process. AhR's transactivation domain might also interact with chromatin remodeling factors and/or histone acetyltransferases to mediate TCDD-induced, AhR/Arnt dependent changes in chromatin structure.

Recent Publications:

Okino ST, Whitlock JP Jr
Dioxin induces localized, graded changes in chromatin structure: implications for Cyp1A1 gene transcription.
Mol Cell Biol. 1995 Jul; 15(7): 3714-3721.

Ko HP, Okino ST, Ma Q, Whitlock JP Jr
Dioxin-induced CYP1A1 transcription in vivo: the aromatic hydrocarbon receptor mediates transactivation, enhancer-promoter communication, and changes in chromatin structure.
Mol Cell Biol. 1996 Jan; 16(1): 430-436.

Ko HP, Okino ST, Ma Q, Whitlock JP Jr
Transactivation domains facilitate promoter occupancy for the dioxin-inducible CYP1A1 gene in vivo.
Mol Cell Biol. 1997 Jul; 17(7): 3497-3507.


Protein-Protein Interactions

Protein-protein interactions play important roles in the induction of gene transcription by TCDD. For example, interactions between the bHLH/PAS proteins AhR and Arnt generate a heterodimer that binds to multiple sites on a xenobiotic-responsive enhancer (XRE) upstream of the CYP1A1 gene, thereby producing a change in chromatin structure and activating transcription. AhR's transactivation domain facilitates occupancy of the CYP1A1 promoter, probably by recruiting a general transcription factor(s) to the promoter via protein-protein interactions. Other factors, such as a cytoplasmic AhR interacting protein and a labile repressor protein, may also influence AhR and Arnt function. Arnt may also interact with different bHLH/PAS proteins in response to other environmental stimuli such as low oxygen tension, thereby generating a heterodimer that regulates the transcription of hypoxia-responsive genes.

Recent Publications:

Ko HP, Okino ST, Ma Q, Whitlock JP Jr
Dioxin-induced CYP1A1 transcription in vivo: the aromatic hydrocarbon receptor mediates transactivation, enhancer-promoter communication, and changes in chromatin structure.
Mol Cell Biol. 1996 Jan; 16(1): 430-436.

Li H, Ko HP, Whitlock JP
Induction of phosphoglycerate kinase 1 gene expression by hypoxia. Roles of Arnt and HIF1alpha.
J Biol Chem. 1996 Aug 30; 271(35): 21262-21267. PDF full text reprint

Ma Q, Whitlock JP Jr
A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.
J Biol Chem. 1997 Apr 4; 272(14): 8878-8884. PDF full text reprint

Ko HP, Okino ST, Ma Q, Whitlock JP Jr
Transactivation domains facilitate promoter occupancy for the dioxin-inducible CYP1A1 gene in vivo.
Mol Cell Biol. 1997 Jul; 17(7): 3497-3507.


Hypoxia-Responsive Genes

Arnt participates in the transcriptional responses to two environmental stimuli (dioxin and hypoxia); it may also play a role in other responses that remain to be identified. The HIF1 alpha/Arnt-dependent transcriptional response to hypoxia allows us to analyze the mechanism by which cells adapt to low oxygen tension, thereby increasing our understanding of bHLH/PAS proteins and the regulation of inducible mammalian gene expression.

Recent Publications:

Li H, Ko HP, Whitlock JP
Induction of phosphoglycerate kinase 1 gene expression by hypoxia. Roles of Arnt and HIF1alpha.
J Biol Chem. 1996 Aug 30; 271(35): 21262-21267. PDF full text reprint

Okino ST, Chichester CH, Whitlock JP Jr
Hypoxia-inducible mammalian gene expression analyzed in vivo at a TATA-driven promoter and at an initiator-driven promoter.
J Biol Chem 1998 Sep 11; 273(37): 23837-43.


New Dioxin-Responsive Genes

Studies of the CYP1A1 gene have revealed interesting aspects of dioxin action and the regulation of mammalian gene expression. We also know that the dioxin-responsive system can function in other chromosonal contexts; however, the mechanism(s) underlying this versatility is not well understood. To address this issue, we have identified additional genes that respond to TCDD and have analyzed their regulation. These experiments place AhR/Arnt function is a broader context. In addition, such studies generate a broader perspective of dioxin biology and may provide new insights into dioxin toxicity.

Recent Publications:

Dong L, Ma Q, Whitlock JP Jr
Down-regulation of major histocompatibility complex Q1b gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
J Biol Chem. 1997 Nov 21; 272(47): 29614-29619. PDF full text reprint

Gao L, Dong L, Whitlock JP Jr
A novel response to dioxin. Induction of ecto-ATPase gene expression.
J Biol Chem 1998 Jun 19; 273(25): 15358-65.

 


Dept. of Molecular Pharmacology

Stanford University Medical Center

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Originally created by Felix W. Frueh
Updated and maintained by Caroline Johnston
Updated 7/99