Abstract
Cellular adaptation to hypoxia occurs via a complex programme of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity.
Highlights
When cells experience oxygen deprivation, the highly conserved canonical hypoxia signalling pathway is activated
We have previously investigated the temporal dynamics of both hypoxia-inducible factor (HIF)-1a and HIF-2a at the single-cell level and demonstrated that they both displayed pulsatile dynamics [24]
The non-homogeneous HIF-2a distribution was not unique to HeLa cells and was observed in a range of mammalian cells, including human embryonic kidney and mouse myoblast cells. These results demonstrate that the observed speckle distribution of HIF-2a is not due to artefacts of ectopic expression, presence of a tag 2 or cell type
Summary
When cells experience oxygen deprivation, the highly conserved canonical hypoxia signalling pathway is activated. HIF-1a was discovered in 1992 [1] This was shortly followed by HIF-2a, which was sequenced simultaneously by different laboratories, published under several names: endothelial PAS domain protein-1 (EPAS1), HIF-1a-like factor (HLF) or members of PAS superfamily 2 (MOP2) [2,3,4,5]. This beta-helix– loop–helix (bHLH) protein is similar to HIF-1a in that it can form a heterodimer with HIF-1b, recognize the same DNA consensus sequence (Hypoxia Response Element/HRE) and activate transcription of hypoxia-inducible genes [3]. The expression level and transcriptional activity of HIF-2a has been shown to be regulated by the same oxygen-dependent mechanisms as HIF-1a (i.e. via the hydroxylation of specific prolyl and asparagyl residues [6,7])
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