The Mammalian Hypoxia Response Pathway: Regulation of HIf and HIF Prolyl Hydroxylases



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Cells exposed to hypoxia -limited oxygen availability- initiate an adaptive response orchestrated by a transcription factor called Hypoxia Inducible Factor (HIF). HIF is composed of an oxygen-sensitive alpha -, and an oxygen-insensitive beta -subunit (ARNT). The stability and transcriptional activity of HIF alpha are controlled by two different Fe(II)- and 2- oxoglutarate-dependent dioxygenases that utilize molecular oxygen during hydroxylation of HIF alpha -subunit. When oxygen levels are sufficient (normoxia), HIF Prolyl Hydroxylases (HPH-1, -2, and -3) hydroxylate the Oxygen-dependent Degradation Domain (ODD) of HIF-alpha targeting it to ubiquitin-mediated proteosomal degradation. Factor Inhibiting HIF 1 (FIH-1, an asparaginyl hydroxylase), on the other hand, hydroxylates C-terminal Transactivation Domain (CTAD) thereby abolishing recruitment of transcriptional co-activators by HIF alpha. However, under hypoxic conditions, both hydroxylations are diminished allowing HIF alpha to escape degradation and induce transcription by associating with co-activators. Because of its critical role as an oxygen sensor, we studied HIF Prolyl Hydroxylase 2 (HPH-2) and focused on protein-protein interactions expecting that some of the interacting proteins might regulate its function. We characterized the function of a HPH-2 interacting protein identified in yeast two-hybrid screen; Inhibitor of Growth 4 (ING4) -a candidate tumor suppressor protein-, and showed that ING4 represses HIF transcriptional activity under hypoxia in a chromatindependent manner. Recruitment of ING4 to alter HIF transcriptional activity represents a novel function of HPH-2. To shed some light on the mechanism of this transcriptional repression, we purified ING4 containing co-repressor complex containing MYST2 and JADE3. Furthermore, we showed that ING4 and MYST2 targets not only HIF but also NF- κB transcription factor, a previously identified target of ING4, perhaps misregulation of which in the absence of functional ING4 protein contributes to tumor progression. Moreover, we identified additional HPH-2 interacting proteins and found that HPH enzymes can be modified by Protein Arginine Methyltransferase 1 (PRMT1) in vitro. Inhibition of methyltransferases in vivo further stabilized and activated HIF-1alpha suggesting a role for methyltransferases in regulation of HIF that might be mediated through HPH enzymes. Methylation of HPH enzymes, the first identified post-translational modification of these enzymes, adds another layer of complexity to the regulation of HIF alpha and it may serve as an interface between the hypoxia response pathway and other signaling pathways.