Structure and sequence conservation of a putative hypoxia response element in the lactate dehydrogenase-B gene of Fundulus

Many aquatic habitats are characterized by periodic or sustained episodes of low oxygen concentration, or hypoxia, and organisms that survive in these habitats do so by utilizing a suite of behavioral, physiological and biochemical adjustments to low oxygen (1-3). In the killifish Fundulus heteroclitus, one response to prolonged exposure to hypoxia is an increase in the activity of lactate dehydrogenase-B (LDH-B), the terminal enzyme of anaerobic glycolysis, in liver tissue (4). An increase in glycolytic enzyme activity also occurs in mammalian cells during hypoxia, a process due, in part, to increased rates of gene transcription mediated by the hypoxia-inducible transcription factor, HIF-1 (5). Given that a homolog of HIF-1 has been identified in fish (6), we hypothesized that HIF might be involved in the observed up-regulation of LDH-B in F. heteroclitus. Herein, we describe the presence of DNA elements in intron 2 of the Ldh-B gene from F. heteroclitus that resemble hypoxia response elements (HRE) described for mammalian genes (7-10). Specifically, over a region of approximately 50 base pairs we identified two consensus HIF-1 binding sires, as well as DNA elements that may bind other transcription factors (e.g., cyclic AMP response elements; CRE). We found that these sites were perfectly conserved among geographically diverse populations of F. heteroclitus, as well as being highly conserved among multiple species in the genus Fundulus. The spacing, orientation, and sequence conservation of these putative regulatory elements suggest that they may be functionally involved in the hypoxic regulation of Ldh-B in these fish.