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The asparaginyl hydroxylase FIH [factor inhibiting HIF (hypoxia-inducible factor)] was first identified as a protein that inhibits transcriptional activation by HIF, through hydroxylation of an asparagine residue in the CAD (C-terminal activation domain). More recently, several ARD [AR (ankyrin repeat) domain]-containing proteins were identified as FIH substrates using FIH interaction assays. Although the function(s) of these ARD hydroxylations is unclear, expression of the ARD protein Notch1 was shown to compete efficiently with HIF CAD for asparagine hydroxylation and thus to enhance HIF activity. The ARD is a common protein domain with over 300 examples in the human proteome. However, the extent of hydroxylation among ARD proteins, and the ability of other members to compete with HIF–CAD for FIH, is not known. In the present study we assay for asparagine hydroxylation in a bioinformatically predicted FIH substrate, the targeting subunit of myosin phosphatase, MYPT1. Our results confirm hydroxylation both in cultured cells and in endogenous protein purified from animal tissue. We show that the extent of hydroxylation at three sites is dependent on FIH expression level and that hydroxylation is incomplete under basal conditions even in the animal tissue. We also show that expression of MYPT1 enhances HIF–CAD activity in a manner consistent with competition for FIH and that this property extends to other ARD proteins. These results extend the range of FIH substrates and suggest that cross-competition between ARDs and HIF–CAD, and between ARDs themselves, may be extensive and have important effects on hypoxia signalling. [ABSTRACT FROM AUTHOR]

The transcription factor HIF (hypoxia-inducible factor) mediates a highly pleiotrophic response to hypoxia. Many recent studies have focused on defining the extent of this transcriptional response. In the present study we have analysed regulation by hypoxia among transcripts encoding human Fe(II)- and 2-oxoglutarate-dependent oxygenases. Our results show that many of these genes are regulated by hypoxia and define two groups of histone demethylases as new classes of hypoxia-regulated genes. Patterns of induction were consistent across a range of cell lines with JMJD1A (where JMJD is Jumonji-domain containing) and JMJD2B demonstrating robust, and JMJD2C more modest, up-regulation by hypoxia. Functional genetic and chromatin immunoprecipitation studies demonstrated the importance of HIF-1α in mediating these responses. Given the importance of histone methylation status in defining patterns of gene expression under different physiological and pathophysiological conditions, these findings predict a role for the HIF system in epigenetic regulation. [ABSTRACT FROM AUTHOR]

The activity and levels of the metazoan HIF (hypoxia-inducible factor) are regulated by its hydroxylation, catalysed by 2OG (2-oxoglutarate)- and Fe(II)-dependent dioxygenases. An oxygen consumption assay was developed and used to study the relationship between HIF hydroxylase activity and oxygen concentration for recombinant forms of two human HIF hydroxylases, PHD2 (prolyl hydroxylase domain-containing protein 2) and FIH (factor inhibiting HIF), and compared with two other 2OG-dependent dioxygenases. Although there are caveats on the absolute values, the apparent Km (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent Km (oxygen) values compared with shorter synthetic peptides of HIF. The analyses also suggest that human PHD2 is selective for fragments of the C-terminal over the N-terminal oxygen-dependent degradation domain of HIF-1α. The present results, albeit obtained under non-physiological conditions, imply that the apparent Km (oxygen) values of the HIF hydroxylases enable them to act as oxygen sensors providing their in vivo capacity is appropriately matched to a hydroxylation-sensitive signalling pathway. [ABSTRACT FROM AUTHOR]