REES, B.B.*; GUITERREZ, Y.I.; BECKMAN, B.S.; SCHULTE, P.M.; University of New Orleans, LA; Tulane University, New Orleans, LA; Tulane University, New Orleans, LA; University of British Columbia, Vancouver: Definition of a hypoxia responsive element in the Fundulus heteroclitus Ldh-B promoter

The hypoxia inducible transcription factor (HIF) binds to several mammalian genes at specific DNA sequences (hypoxia responsive elements; HRE) and increases their expression during exposure of cells and tissues to low oxygen. Recently, homologs of HIF have been described in non-mammalian vertebrates, but the genes that are subject to regulation by HIF in these organisms remain unknown. In the killifish, Fundulus heteroclitus, previous research showed that the activity of the liver isozyme of lactate dehydrogenase (LDH-B) is up-regulated during hypoxia and that the Ldh-B gene contains DNA sequences that form consensus HIF binding sites. Here, we evaluated the ability of these putative HREs to drive the expression of reporter gene constructs in transfected cells in culture and in live fish. We found that the promoter region of the Ldh-B gene conferred hypoxia-sensitivity to reporter gene expression in transiently transfected mammalian Hep 3B cells. Through a series of truncations and mutations we defined an HRE consisting of an inverted repeat of GATGTG spaced by 8 base pairs, at between 150 and 250 base pairs upstream of the transcription start site. Preliminary experiments in which live fish were injected with reporter plasmids containing the Ldh-B promoter also showed hypoxia-dependent expression. In the same experiments, however, LDH-B activity did not increase. These results suggest multiple controls on the up-regulation of LDH-B activity in vivo. Perhaps other transcription factors or accessory proteins, acting in concert with HIF, are required for the increase in LDH-B activity during hypoxic exposure of intact fish. Supported by grants from NSF and NSERC.