Characterising mechanisms of regulation of hypoxia-inducible factors.

All humans have a constant and absolute requirement for oxygen, which is necessary to produce energy for normal cell growth and survival. Mammalian cells adapt to low oxygen stress (hypoxia) through a transcriptional response pathway mediated by the Hypoxia-inducible factor protein, HIF. HIF is a heterodimer consisting of one of three alpha subunits (HIF-1a, HIF-2a or HIF-3a) and a beta subunit called Amt. The activation of HIF by hypoxia is a multistep process involving increases in both protein stabilisation and transcriptional potency of the alpha subunits. Protein stabilisation is a result of inhibition of ubiquitin dependent degradation while increased transactivation is a result of recruitment of transcriptional coactivators. Once activated the HIF alpha subunits accumulate in the nucleus where they heterodimerise with Arnt to bind hypoxia response elements (HRE) located in genes involved in helping cells adapt to low oxygen stress. To better understand the mechanisms by which HIF is regulated I have undertaken in my PhD a study to investigate both the DNA binding and transcriptional capacity of the HIF-1a, and HIF-2a subunits. In my thesis I report that the DNA binding of HIF-2a, to a HRE, but not HIF-1a, is dependent on redox reducing conditions. In-vitro DNA binding and mammalian two-hybrid assays demonstrate that a unique cysteine residue located in the DNA binding basic region of HIF-2a is a target for the reducing activity of redox factor Ref-1. Our data are the first to establish a discriminating control mechanism for differential regulation of HIFs that targets the DNA binding potential of HIF-2a. In a separate investigation we show that the induction of the hypoxia sensitive HIFa carboxy-terminal transactivation domain (CAD) occurs through abrogation of hydroxylation of a conserved asparagine residue. Hypoxia and chemical mimetics of hypoxia, such as iron chelators and analogs of 2-oxoglutarate, prevented hydroxylation of the asparagine allowing the CAD to interact with the transcriptional coactivator protein p300. We then demonstrate that the protein factor inhibiting HIF-1 (FIH-1), previously shown to interact with HIF, is an asparaginyl hydroxylase enzyme capable of hydroxylating the asparagine in the HIF CAD. Like other known hydroxylases FIH-1 is an iron and 2- oxoglutarate-dependent enzyme that uses molecular oxygen to modify its substrate, thus comprising a critical regulatory component of the oxygen sensing and HIF response pathway.

Author dc:creator

• Lando, David

Advisors dc:contributor.advisor

• Whitelaw, Murray
• Peet, Dan

Handle dc:identifier.uri

http://hdl.handle.net/2440/99907

OAI identifier oai:identifier

oai:digital.library.adelaide.edu.au:2440/99907