Dimerization, DNA Binding, and Transactivation Properties of Hypoxia-inducible Factor 1

doi:10.1074/jbc.271.30.17771

Dimerization, DNA Binding, and Transactivation Properties of Hypoxia-inducible Factor 1*

From the Center for Medical Genetics, Departments of Pediatrics and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914

^‡ Established Investigator of the American Heart Association. To whom correspondence should be addressed:
The Johns Hopkins Hospital, CMSC-1004, 600 N. Wolfe St., Baltimore, MD 21287-3914.
Tel.: 410-955-1619; Fax: 410-955-0484. E-mail: gsemenza{at}welchlink.welch.jhu.edu

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes including the human erythropoietin (EPO) gene. In this study, we report structural features of the HIF-1α subunit that are required for heterodimerization, DNA binding, and transactivation. The HIF-1α and HIF-1β (ARNT; aryl hydrocarbon receptor nuclear translocator) subunits were coimmunoprecipitated from nuclear extracts, indicating that these proteins heterodimerize in the absence of DNA. In vitro-translated HIF-1α and HIF-1β generated a HIF-1/DNA complex with similar electrophoretic mobility and sequence specificity as HIF-1 present in nuclear extracts from hypoxic cells. Compared to 826-amino acid, full-length HIF-1α, amino acids 1-166 mediated heterodimerization with HIF-1β (ARNT), but amino acids 1-390 were required for optimal DNA binding. A deletion involving the basic domain of HIF-1α eliminated DNA binding without affecting heterodimerization. In cotransfection assays, forced expression of recombinant HIF-1α and HIF-1β (ARNT) activated transcription of reporter genes containing EPO enhancer sequences with intact, but not mutant, HIF-1 binding sites. Deletion of the carboxy terminus of HIF-1α (amino acids 391-826) markedly decreased the ability of recombinant HIF-1 to activate transcription. Overexpression of a HIF-1α construct with deletions of the basic domain and carboxy terminus blocked reporter gene activation by endogenous HIF-1 in hypoxic cells.

Footnotes

↵* This work was supported by grants from the American Heart Association, Lucille P. Markey Charitable Trust, and National Institute of Diabetes, Digestive and Kidney Diseases (R01-DK39869). 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:

EPO

erythropoietin

bp

base pair(s)

HIF-1

hypoxia-inducible factor 1

AHR

aryl hydrocarbon receptor

ARNT

AHR nuclear translocator

bHLH

basic helix-loop-helix

PAS

PER-ARNT-SIM

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

IP

immunoprecipitation

EMSA

electrophoretic mobility shift assay

CAT

chloramphenicol acetyltransferase

βgal

β-galactosidase

FL

full length.
↵² B.-H. Jiang and G. L. Semenza, unpublished data.
- Received February 1, 1996.
- Revision received April 19, 1996.