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Molecular and Cellular Biology, June 2002, p. 4319-4333, Vol. 22, No. 12
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.12.4319-4333.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Recruitment of the NCoA/SRC-1/p160 Family of Transcriptional Coactivators by the Aryl Hydrocarbon Receptor/Aryl Hydrocarbon Receptor Nuclear Translocator Complex

Timothy V. Beischlag,1,{dagger} Song Wang,1 David W. Rose,2 Joseph Torchia,3 Suzanne Reisz-Porszasz,1,{ddagger} Khurshid Muhammad,1 Walter E. Nelson,1 Markus R. Probst,1,3 Michael G. Rosenfeld,4 and Oliver Hankinson1*

Department of Pathology and Laboratory Medicine, Molecular Biology Institute, and Jonsson Comprehensive Cancer Center, School of Medicine, University of California—Los Angeles, Los Angeles, California 90095,1 Division of Endocrinology and Metabolism,2 Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093 ,4 Departments of Pharmacology and Toxicology and of Oncology, Regional London Cancer Centre, University of Western Ontario, London, Ontario, Canada3

Received 8 November 2001/ Returned for modification 12 December 2001/ Accepted 11 March 2002

The aryl hydrocarbon receptor complex heterodimeric transcription factor, comprising the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) domain aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, mediates the toxic effects of TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin). The molecular events underlying TCDD-inducible gene activation, beyond the activation of the AHRC, are poorly understood. The SRC-1/NCoA-1, NCoA-2/GRIP-1/TIF-2, and p/CIP/AIB/ACTR proteins have been shown to act as mediators of transcriptional activation. In this report, we demonstrate that SRC-1, NCoA-2, and p/CIP are capable of independently enhancing TCDD-dependent induction of a luciferase reporter gene by the AHR/ARNT dimer. Furthermore, injection of anti-SRC-1 or anti-p/CIP immunoglobulin G into mammalian cells abolishes the transcriptional activity of a TCDD-dependent reporter gene. We demonstrate by coimmunoprecipitation and by a reporter gene assay that SRC-1 and NCoA-2 but not p/CIP are capable of interacting with ARNT in vivo after transient transfection into mammalian cells, while AHR is capable of interacting with all three coactivators. We confirm the interactions of ARNT and AHR with SRC-1 with immunocytochemical techniques. Furthermore, SRC-1, NCoA-2, and p/CIP all associate with the CYP1A1 enhancer region in a TCDD-dependent fashion, as demonstrated by chromatin immunoprecipitation assays. We demonstrate by yeast two-hybrid, glutathione S-transferase pulldown, and mammalian reporter gene assays that ARNT requires its helix 2 domain but not its transactivation domain to interact with SRC-1. This indicates a novel mechanism of action for SRC-1. SRC-1 does not require its bHLH-PAS domain to interact with ARNT or AHR, but utilizes distinct domains proximal to its p300/CBP interaction domain. Taken together, these data support a role for the SRC family of transcriptional coactivators in TCDD-dependent gene regulation.

* Corresponding author. Mailing address: Dept. of Pathology and Laboratory Medicine, UCLA Medical Center, Center for Health Sciences, Box 951732, Los Angeles, CA 90095-1732. Phone: (310) 825-2936. Fax: (310) 794-9272. E-mail: ohank{at}mednet.ucla.edu.

{dagger} Present address: Dept. of Medicine, University of California, San Diego, La Jolla, CA 92093-0648.

{ddagger} Present address: Division of Endocrinology, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059.

§ Present address: Basilia Pharmaceutica Ltd., CH-4002 Basel, Switzerland.

Molecular and Cellular Biology, June 2002, p. 4319-4333, Vol. 22, No. 12
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.12.4319-4333.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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