DNA Binding Specificities and Pairing Rules of the Ah Receptor, ARNT, and SIM Proteins (*)
- From the Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611
- § To whom correspondence should be addressed: Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL, 60611. Tel.: 312-503-9855; Fax: 312-503-5349; c-bradfield{at}nwu.edu.
Abstract
The Ah receptor (AHR), the Ah receptor nuclear translocator protein (ARNT), and single-minded protein (SIM) are members of the basic helix-loop-helix-PAS (bHLH-PAS) family of regulatory proteins. In this study, we examine the DNA half-site recognition and pairing rules for these proteins using oligonucleotide selection-amplification and coprecipitation protocols. Oligonucleotide selection-amplification revealed that a variety of bHLH-PAS protein combinations could interact, with each generating a unique DNA binding specificity. To validate the selection-amplification protocol, we demonstrated the preference of the AHR•ARNT complex for the sequence commonly found in dioxin-responsive enhancers in vivo (TNGCGTG). We then demonstrated that the ARNT protein is capable of forming a homodimer with a binding preference for the palindromic E-box sequence, CACGTG. Further examination indicated that ARNT may have a relaxed partner specificity, since it was also capable of forming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG. Coprecipitation experiments using various PAS proteins and ARNT were consistent with the idea that the ARNT protein has a broad range of interactions among the bHLH-PAS proteins, while the other members appear more restricted in their interactions. Comparison of this in vitro data with sites known to be bound in vivo suggests that the high affinity half-site recognition sequences for the AHR, SIM, and ARNT are T(C/T)GC, GT(G/A)C (5′-half-sites), and GTG (3′-half-sites), respectively.
Footnotes
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↵* This work was supported by The Pew Foundation, National Institutes of Health Grants ES-05703, ES-05660, and ES-05589, and a postdoctoral fellowship sponsored by The Colgate-Palmolive Co. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- AHR
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Ah receptor
- bHLH
-
basic helix-loop-helix
- TCDD
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2,3,7,8-tetrachlorodibenzo-p-dioxin
- DRE
-
dioxin-responsive elements
- PER
-
period
- Hsp90
-
heat shock protein of 90 kDa
- AHRCΔ516
-
AHR deletion in which 516 amino acid residues have been removed from the C terminus
- AHRGNΔ315
-
AHR construct in which the N-terminal 316 amino acids have been replaced by the DNA binding and dimerization domain of Gal4(3)
- PCR
-
polymerase chain reaction.
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↵2 In this manuscript, we refer to the DNA binding complexes of bHLH-PAS proteins as dimers. This assumption is based on x-ray crystallographic evidence demonstrating that the fundamental DNA binding complex of most bHLH proteins is dimeric(23, 24, 49). However, we cannot rule out the possibility that higher order complexes exist, such as dimers of dimers (tetramers), with each dimer independently interacting with separate DNA sites(22). The existence of such higher order complexes would not alter any of our conclusions.
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↵3 Bona fide enhancer elements are those defined DREs that have been shown to regulate gene transcription in a ligand-responsive manner in vivo.
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↵4 Interestingly, this consensus sequence did not contain a C at position 4 as was observed in the analysis presented in Fig. 1C. Given the proximity of this C to the primer site in OL187, the observed bias reported using that oligonucleotide, and our inability to reproduce the conservation of C at position 4 using OL224, we consider the assignments derived in Fig. 2C as our final AHR•ARNT selected consensus sequence.
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- Received May 22, 1995.
- Revision received August 24, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
