Bacterially produced human HIF-1a is competent for heterodimerization and specific DNA-binding Georgia Chachami a , Efrosyni Paraskeva b , Eleni Georgatsou a , Sofia Bonanou a , George Simos a, * a Laboratory of Biochemistry, School of Medicine, University of Thessaly, Papakyriazi 22, 41222 Larissa, Greece b Laboratory of Physiology, School of Medicine, University of Thessaly, Papakyriazi 22, 41222 Larissa, Greece Received 28 March 2005 Available online 6 April 2005 Abstract Hypoxia-inducible factor 1a (HIF-1a) is the regulatory subunit of HIF-1, the transcriptional activator and key mediator of the cellular response to hypoxia. Regulation of HIF-1a occurs at multiple levels and involves several different post-translational mod- ifications. In order to examine the importance of these modifications for the basic function of HIF-1a, we have produced in bacteria recombinant full-length human HIF-1a using different expression systems. We show that this unmodified form of HIF-1a is able to form a stable heterodimer with the second subunit of HIF-1 (HIF-1b or ARNT) when both proteins are co-expressed in Escherichia coli. Furthermore, this bacterially reconstituted heterodimer exhibits specific DNA-binding activity. These data indicate that post- translational modification of HIF-1a is not essential for its interaction with ARNT and DNA, and provide an in vitro system for the characterization of the molecular properties of HIF-1a. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Hypoxia-inducible factor 1; HIF-1a; ARNT; Bacterial expression; Heterodimerization; DNA-binding Upon hypoxia (reduced oxygen availability), cells re- spond by increasing the expression of genes encoding proteins that facilitate oxygen delivery (e.g., erythropoi- etin, VEGF) or enhance anaerobic production of energy (e.g., glycolytic enzymes) thus maintaining energy homeostasis [1]. A crucial component in the induction of hypoxia-regulated genes is the transcription factor hypoxia-inducible factor 1 (HIF-1), which activates transcription by binding to a specific cis-acting regula- tory sequence referred to as hypoxia response element (HRE), a hallmark of hypoxia-sensitive target genes [2]. The HIF-1 protein complex consists of a heterodi- mer composed of HIF-1a and HIF-1b subunits, both members of the basic-helix–loop–helix–PAS protein family of transcription factors [3,4]. HIF-1a is unique to HIF-1 while HIF-1b is identical to the aryl hydrocar- bon receptor nuclear translocator (ARNT) that also dimerizes with the aryl hydrocarbon receptor. Although HIF-1b is constitutively expressed, the activity of HIF-1a is regulated at multiple levels [5]. Under normoxic conditions, HIF-1a is maintained at a low level through proteasomal degradation. HIF-1a is targeted for proteolysis through its interaction with the von Hippel-Lindau (VHL) tumor suppressor protein and subsequent polyubiquitination [6,7]. VHL can only bind to HIF-1a when one or possibly two critical proline residues in the ODD (oxygen dependent degradation) domain of HIF-1a are hydroxylated [8–10]. The proline hydroxylases that modify HIF-1a (members of the EGLN family, termed PHDs or HPHs) are Fe 2+ depen- dent and require oxygen [11]. According to these find- ings, it has been suggested that hypoxia inhibits the hydroxylation of HIF-1a thus allowing its stabilization 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.193 * Corresponding author. Fax: +30 2410 565054. E-mail address: simos@med.uth.gr (G. Simos). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 331 (2005) 464–470 BBRC