Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively
Authors: Christopher J Schofield
Shan Zhong
Xin Lu
Peter J Ratcliffe
Yihua Wang
Journal: Journal of Cell Science (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: FIH; 2-OG; 2-Oxoglutarate; Dioxygenase inhibitors; Factor inhibiting HIF
Affiliations: Ludwig Institute for Cancer Research Ltd., Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom., Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom., Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom., Ludwig Institute for Cancer Research Ltd., Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom  ., Target Discovery Institute, NDM Research Building, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, United Kingdom.

Abstract:  Hypoxia plays a crucial role at cellular and physiological levels in all animals. The responses to chronic hypoxia are, at least substantially, orchestrated by activation of the hypoxia inducible transcription factors (HIFs), whose stability and subsequent transcriptional activation are regulated by HIF hydroxylases. Factor inhibiting HIF (FIH), initially isolated as a HIFα interacting protein following a yeast two-hybrid screen, is an asparaginyl hydroxylase that negatively regulates transcriptional activation by HIF. This study aimed to define the mechanisms that govern transitions of FIH between the nucleus and cytoplasm. We report that FIH accumulates in the nucleus within a short time window during hypoxia treatment. We provide evidence, based on the application of genetic interventions and small molecule inhibition of the HIF hydroxylases, that the nuclear localization of FIH is governed by two opposing processes: nuclear entry by 'coupling' with HIF1α for importin β1-mediated nuclear import and active export via a Leptomycin B-sensitive exportin1-dependent pathway.This article has an associated First Person interview with the first author of the paper. 

Retracted: No

PubPeer comments:
 Looks like there may be mycoplasma contamination. Could the authors please retest their cell lines. One example is shown from Figure 3A. ![file] ![file] ![file] , 
 Thank you for your enquiries. I am going through each of these carefully both within my own laboratory and, in the case of collaborative work with any other laboratory that was involved. This will take a little time, but I will endeavour to get back with answers to your questions as soon as is possible. , 
 Thank you for your enquiry. At the time of this experiment, we were regularly testing cell lines for mycoplasma, but we cannot rule out the possibility of mycoplasma infection in this particular experiment. Given the time that has passed since the date of the experiment, it is not possible to re-test the specific cell line batch that was used. You have based your question about mycoplasma contamination on the presence of DAPI-stained material outside the nucleus in these microscopy images. We have reviewed the literature and it should be noted that extranuclear DAPI staining is not by itself a reliable indicator of mycoplasma infection (for example, [Nature Protocols](http: www.nature.com articles nprot.2010.43) 5, 929u2013934(2010) https: www.nature.com articles nprot.2010.43). This is due to the fact that the results are difficult to interpret as many cellular manipulations can also cause small DNA staining. While we canu2019t rule out mycoplasma contamination, it is equally difficult to be certain that the signals were from mycoplasma using the image data alone. However, even if these cells were potentially contaminated with mycoplasma, we do not think that this has affected the result. The result from this circled panel in the figure u2013 that DMOG induces nuclear localisation of HIF1u03b1 u2013 is a positive control and has been replicated by others, for example Salvi Thanabalu, 2016 (PMID: 28955984). Our result matches those elsewhere in the literature. To remove any doubt, we have repeated the experiment using the same cell type (U2OS) as the original experiment: ![file] These cell lines tested negative for mycoplasma using the MycoAlertTM mycoplasma detection kit (LONZA) in the same week as repeating the experiment. As you can see, we have reproduced our original finding that siRNA-dependent depletion of importinu03b21 blocked the nuclear accumulation of HIF1u03b1 and FIH induced by DMOG treatment. We are therefore confident in this result. The raw data from this experiment is available from [Mendeley data](http: dx.doi.org 10.17632 gvzn7577jk.1) (http: dx.doi.org 10.17632 gvzn7577jk.1). While preparing this response, we also observed that several panels in Figure 4C have bright TO-PRO-3-stained puncta across the image that are not restricted to within cells. Given the size and brightness of these dots, and the fact that they are also present outside cells, we do not consider that it is likely that they represent mycoplasma that was undetected by our testing. A plasmid transfection was performed as part of this experiment and so we consider these dots most likely to be leftover transfection material. As a matter of course, we have informed the Journal of this correspondence. , 
 #1 DAPI will also stain mitochondrial DNA, and it is not uncommon to see cytoplasmic DAPI staining if the concentration used is high enough, or the laser power is high enough. In my extensive experience as a mitochondrial biologist, the staining in those images looks very much like mitochondria. ,