A novel hypoxia-response element in the lactate dehydrogenase-B gene of the killifish Fundulus heteroclitus Bernard B. Rees a, ⁎, Yanira G. Figueroa b , Thomas E. Wiese c,d , Barbara S. Beckman b,d , Patricia M. Schulte e a Department of Biological Sciences, University of New Orleans, New Orleans, LA, 70148, USA b Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA, 70112, USA c College of Pharmacy, Xavier University of Louisiana, New Orleans, LA, 70125, USA d Tulane/Xavier Center for Bioenvironmental Research, New Orleans, LA, 70112, USA e Department of Zoology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 abstract article info Article history: Received 9 March 2009 Received in revised form 4 May 2009 Accepted 5 May 2009 Available online 8 May 2009 Keywords: Cell culture Gene expression Hypoxia-inducible factor Lactate dehydrogenase Mummichog Oxygen Teleost Previous studies have suggested that the lactate dehydrogenase-B gene (Ldh-B) of the Atlantic killifish, Fundulus heteroclitus, is a hypoxia-responsive gene. Here, we demonstrate that the F. heteroclitus Ldh-B promoter confers hypoxia-dependence upon reporter gene expression in transiently transfected mammalian (Hep3B) and fish (RTG-2 and RTH-149) cells in culture. Mutation and deletion analyses identified a putative hypoxia-response element (HRE) between 109 and 90 nucleotides upstream of the major start site. This HRE is characterized by the sequence 5′-GATGTG-3′ spaced by 8 nucleotides from a perfect inverted repeat, and both sites are necessary for hypoxic induction of reporter gene expression in mammalian and fish cells. This HRE differs from the canonical sequence at one nucleotide position that is invariant among HREs from a wide range of hypoxia-sensitive genes. In fish cells, maximal induction of reporter gene expression driven by this HRE occurred at the lowest oxygen level tested (0.5%), took 48 h to 96 h, and was independent of glucose concentration (between 5.6 and 25 mM). Under all conditions tested, hypoxic induction of gene expression was lower in RTH-149 cells than in RTG-2, suggesting a potential defect in hypoxia signaling in RTH-149 cells. These results demonstrate that the F. heteroclitus Ldh-B promoter contains a novel HRE that is capable of driving reporter gene expression in a sequence-specific and oxygen-, time-, and cell line-dependent manner. © 2009 Elsevier Inc. All rights reserved. 1. Introduction In animals, low oxygen (hypoxia) promotes a variety of physiolo- gical and biochemical responses that improve tissue oxygen delivery or cellular hypoxia tolerance. The molecular bases of these responses have been best studied in mammals, in which the hypoxia-inducible transcription factors (HIF) play a key role. This family of transcription factors is comprised of multiple α subunits (HIF-1α, HIF-2α, and HIF- 3α), which accumulate during hypoxia, dimerize with the β subunit (HIF-β, previously described as the aryl hydrocarbon receptor nuclear translocator or ARNT), bind to regulatory regions of target genes, and alter rates of gene expression. More than 70 genes are regulated by HIF, including genes involved in vasculogenesis, erythropoiesis, glucose uptake and metabolism, iron and catecholamine metabolism, and a variety of other cellular processes (Rocha, 2007; Kaluz et al., 2008). The HIF dimer interacts with its target genes at the hypoxia- response element (HRE). A survey of HREs from hypoxia-responsive genes revealed a conserved sequence of RCGTG (where R is A or G) that is necessary for HIF binding and hypoxic induction of gene expression (Wenger et al., 2005; Kaluz et al., 2008). In addition to the HIF-binding site, most functional HREs include additional nearby protein binding sites. For example, a cAMP-responsive element (CRE) downstream of the HIF-binding site is necessary for hypoxia- responsive transcription of Ldh-A (Firth et al., 1995), the sequence CACAG is critical to the function of the erythropoietin and transferrin HREs (Semenza and Wang, 1992; Lok and Ponka, 1999), and a HIF- ancillary sequence (HAS) is necessary for hypoxic induction of vascular endothelial growth factor (VEGF) and other mammalian genes (Kimura et al., 2001). Many fish and other aquatic organisms occur in environments characterized by spatial and temporal fluctuations in oxygen con- centration, frequently including conditions of severe hypoxia (Diaz, 2001). Recent studies on the molecular responses to hypoxia in fish reveal several similarities with their mammalian counterparts (Nikinmaa and Rees, 2005). Multiple forms of HIF-α and ARNT have been described (Soitamo et al., 2001; Powell and Hahn, 2002; Law et al., 2006; Sollid et al., 2006; Rahman and Thomas, 2007; Rytkönen et al., 2007; Terova et al., 2008), and hypoxia leads to widespread changes in gene expression, including up-regulation of evolutionarily conserved genes and processes (Gracey et al., 2001; Ton et al., 2003; van der Meer et al., 2005). At present, though, a functional interaction between HIF and the HRE of a target gene has been described for only Comparative Biochemistry and Physiology, Part A 154 (2009) 70–77 ⁎ Corresponding author. Tel.: +1 504 280 6729; fax: +1 504 280 6121. E-mail address: brees@uno.edu (B.B. Rees). 1095-6433/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2009.05.001 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa