When it comes to surviving hypoxia, or low oxygen, animals get by with a little help from their factors – hypoxia-inducible factors (HIFs), that is. HIFs are useful molecules that, when activated by hypoxia, help form switches to crank gene expression up or down as needed, thereby regulating cellular processes that help animals cope with the stress. Because of the low capacity of water for dissolved oxygen, fish are especially likely to encounter this stress, particularly in shallow, productive environments like estuaries and salt marshes; therefore, estuarine fish, particularly the hypoxia-hardy mummichog (Fundulus heteroclitus), are useful models for studying HIFs. In general, HIF molecules are made of two subunits: the β-subunit, which is always expressed and available, and one of multiple varieties of the α-subunit. One variety, HIF2αa, has already been described in the mummichog, but Ian Townley and his colleagues at the University of New Orleans, USA, as well as researchers from Xavier University of New Orleans, USA, and Woods Hole Oceanographic Institution, USA, sought to discover what other forms of HIFα exist in this fish.
To hunt for HIFαs, Townley and the other researchers used a small piece of a HIF1α gene from rainbow trout and a piece of what they suspected to be a HIF3α gene from the mummichog to probe mummichog liver DNA for matching sequences. This enabled them to determine the full mummichog HIFα sequences and compare them with HIFα genes in other vertebrates. They also investigated in which of the fish's organs the different forms of HIFα were expressed. Finally, they used cultured cells in vitro to translate the HIFα coding sequences into proteins and looked at how well the proteins performed their jobs, such as binding to areas of DNA called hypoxia response elements (HREs) in order to influence gene expression. In this case, the researchers analyzed expression of a specially engineered reporter gene containing mummichog HREs that produces light when activated; this made it easy to detect and quantify HIFα-induced activation by measuring light production.
The team discovered DNA sequences for HIF1α and HIF3α genes in the mummichog that were similar to those of other fish and encoded proteins that functioned as HIFαs should, confirming that mummichog HIFαs operate under the same basic framework as those in other species. They also found a shorter version of HIF2α, HIF2αb, whose sequence suggests it is unable to detect hypoxia or activate genes; alternatively, it may be involved in regulating other HIFαs, similar to one variety of HIF3α found in mammals. The group further noticed that the three full-length forms of HIFα were distributed among tissues in patterns similar to those in other fish. Surprisingly, however, HIFα expression was very low in the brain and heart compared with reported values for other fish species, with unclear implications for HIFα-mediated processes. Furthermore, there were several areas where the sequence was variable in the newly identified HIFα genes as well as the previously identified HIF2αa gene, which could allow for variation in hypoxia responses among individuals or populations.
Many questions remain about exactly how the expression patterns and sequence variations might translate into protein abundance and function, as well as which HIFα characteristics might influence the mummichog's hardiness. Nevertheless, this characterization of mummichog HIFα genes lays important groundwork for further research into why these water warriors are so good at surviving in low-oxygen environments.