The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels.

Anna L Miles,James A Nathan,Stephen P Burr,Guinevere L Grice

doi:10.7554/elife.22693

Abstract

Hypoxia Inducible transcription Factors (HIFs) are principally regulated by the 2-oxoglutarate and Iron(II) prolyl hydroxylase (PHD) enzymes, which hydroxylate the HIFα subunit, facilitating its proteasome-mediated degradation. Observations that HIFα hydroxylation can be impaired even when oxygen is sufficient emphasise the importance of understanding the complex nature of PHD regulation. Here, we use an unbiased genome-wide genetic screen in near-haploid human cells to uncover cellular processes that regulate HIF1α. We identify that genetic disruption of the Vacuolar H+ ATPase (V-ATPase), the key proton pump for endo-lysosomal acidification, and two previously uncharacterised V-ATPase assembly factors, TMEM199 and CCDC115, stabilise HIF1α in aerobic conditions. Rather than preventing the lysosomal degradation of HIF1α, disrupting the V-ATPase results in intracellular iron depletion, thereby impairing PHD activity and leading to HIF activation. Iron supplementation directly restores PHD catalytic activity following V-ATPase inhibition, revealing important links between the V-ATPase, iron metabolism and HIFs.

Highlights

Hypoxia Inducible transcription Factors (HIFs) are major transcriptional regulators of cellular responses to oxygen availability, promoting several metabolic adaptations to ensure cell survival
We developed a forward genetic screening approach to identify genes involved in the regulation of HIF1a under aerobic conditions using near haploid human KBM7 cells expressing a HIF1a-specific fluorescent reporter (HIF1a-GFPODD) (Burr et al, 2016)
This screen involved randomly mutagenising a clonal population of KBM7 HIF1a-GFPODD reporter cells with a gene-trapping retrovirus, fluorescence activated cell sorting (FACS) to enrich for rare mutations that resulted in increased GFP expression, and mapping the insertion sites in these GFPHIGH cells with Illumina HiSeq

Summary

Introduction

HIFs are major transcriptional regulators of cellular responses to oxygen availability, promoting several metabolic adaptations to ensure cell survival. HIF1a (the ubiquitously expressed HIFa isoform) is a very short-lived protein (Berra et al, 2001), and the efficiency of VHL in promoting proteasomal degradation has led to the recent development of small molecules that hijack the VHL complex to selectively destroy target proteins as a potential therapeutic tool (Bondeson et al, 2015). Despite this clear role for proteasomal degradation of HIF, it has been reported that lysosomal inhibitors can lead to stabilisation of the HIFa subunit in both normal oxygen levels and in hypoxia. This stabilisation can lead to a functional HIF response (Lim et al, 2006), and upregulation of target genes to promote glucose metabolism and angiogenesis (Hubbi et al, 2013)

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