Abstract
Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia.
Highlights
Recent work has revealed an extensive family of Fe(II) and 2oxoglutarate (2OG) dependent oxygenases encoded in the human genome
ALKBH5 is a hypoxia-induced gene To determine the extent of regulation by hypoxia among the human ALKBH family, we exposed a series of cell lines to hypoxia and tested for alterations in transcript levels by RT-Q-PCR across all ALKBH family members
No enrichment was observed in DNA encompassing HRE B. (E) MCF7 cells were transfected with pGL3promoter vectors expressing the putative ALKBH5 HRE A sequence. pGL3promoter vectors that were either ‘‘empty’’ or contained the CA9 HRE served as controls as did an ‘‘irrelevant’’ vector
Summary
Recent work has revealed an extensive family of Fe(II) and 2oxoglutarate (2OG) dependent oxygenases encoded in the human genome (reviewed in [1]). Human homologues of the Escherichia coli DNA repair enzyme AlkB are termed ALKBH enzymes. Some ALKBH enzymes have been demonstrated to function as nucleic acid demethylases, catalyzing the oxidative demethylation of 1-methyladenine and 3methylcytosine in DNA and RNA [3,6,7]. Eight human AlkB homologues (ALKBH1-8) have been predicted, of which three (ALKBH1-3) have been shown to exhibit nucleic acid demethylation activity [8,9,10]. A DNA lyase activity has been recently described for ALKBH1 that is Fe(II) and 2OG independent [11]. A tRNA methyltransferase activity of ALKBH8 has been described and implicated in translational decoding [12,13,14]
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