Two Murine Homologs of the Drosophila Single-minded Protein That Interact with the Mouse Aryl Hydrocarbon Receptor Nuclear Translocator Protein

doi:10.1074/jbc.272.7.4451

Two Murine Homologs of the Drosophila Single-minded Protein That Interact with the Mouse Aryl Hydrocarbon Receptor Nuclear Translocator Protein*

From the ^∥ Department of Pathology and Laboratory Medicine, Molecular Biology Institute, California 90095 and
^‡ Jonsson Comprehensive Cancer Center, School of Medicine, UCLA, Los Angeles, California 90095 and the
^§ Department of Anatomy, School of Medicine, University of California, San Francisco, California 94143

** To whom correspondence should be addressed:
Dept. of Pathology and Laboratory Medicine, UCLA Medical Center, Center for the Health Sciences, Box 951732, Los Angeles, CA 90095-1732
. Tel.: 310-825-2936; Fax: 310-794-9272; E-mail: ohank{at}pathology.medsch.ucla.edu.

↵^¶ Present address: Carnegie Institution of Washington, Dept. of Embryology, 115 W. University Pkwy., Baltimore, MD 21210.

Abstract

Drosophila single-minded, which acts as a positive master gene regulator in central nervous system midline formation in Drosophila, its two mouse homologs SIM1 and SIM2, and the mammalian aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins are members of the basic-helix-loop-helix·PAS family of transcription factors. In the yeast two-hybrid system, we demonstrate strong constitutive interaction of ARNT with SIM1 and SIM2 and fully ligand-dependent interaction of ARNT with AHR. Both the helix-loop-helix and the PAS regions of SIM1 and of ARNT are required for efficient heterodimerization. SIM1 and SIM2 do not form homodimers, and they do not interact with AHR. We also failed to detect homodimerization of ARNT.

The interaction of ARNT with SIM1 was confirmed with in vitro synthesized proteins. Like AHR, in vitro synthesized SIM1 associates with the 90-kDa heat shock protein. SIM1 inhibits binding of the AHR·ARNT dimer to the xenobiotic response element in vitro Introduction of SIM1 into hepatoma cells inhibits transcriptional transactivation by the endogenous AHR·ARNT dimer. The mouse SIM1·ARNT dimer binds only weakly to a proposed DNA target for the Drosophila SIM·ARNT dimer. In adult mice mRNA for SIM1 was expressed in lung, skeletal muscle, and kidney, whereas the mRNA for SIM2 was found in the latter two. ARNT is also expressed in these organs. Thus mouse SIM1 and SIM2 are novel heterodimerization partners for ARNT in vitro, and they may function both as positive and negative transcriptional regulators in vivo, during embryogenesis and in the adult organism.

Footnotes

↵* This work was supported by NCI, National Institutes of Health, Grant CA 28868 and by a fellowship from the Swiss National Science Foundation (to M. R. P). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

bHLH

basic-helix-loop-helix

AHR

aryl hydrocarbon receptor

ARNT

aryl hydrocarbon nuclear translocator

BNF

β-naphthoflavone

CAT

chloramphenicol acetyltransferase

CME

central nervous system midline element

SIM

single-minded

EMSA

electrophoretic mobility shift assay

HSP90

90-kDa heat shock protein

PAS

PER-ARNT-SIM homology region

PCR

polymerase chain reaction

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

XRE

xenobiotic response element

kb

kilobase pair(s).
↵² J. Colicelli, personal communication.
- Received May 8, 1996.
- Revision received November 1, 1996.