The Caenorhabditis elegans rhy-1 Gene Inhibits HIF-1 Hypoxia-Inducible Factor Activity in a Negative Feedback Loop That Does Not Include vhl-1

Abstract

Hypoxia-inducible factor (HIF) transcription factors implement essential changes in gene expression that enable animals to adapt to low oxygen (hypoxia). The stability of the C. elegans HIF-1 protein is controlled by the evolutionarily conserved EGL-9/VHL-1 pathway for oxygen-dependent degradation. Here, we describe vhl-1-independent pathways that attenuate HIF-1 transcriptional activity in C. elegans. First, the expression of HIF-1 target genes is markedly higher in egl-9 mutants than in vhl-1 mutants. We show that HIF-1 protein levels are similar in animals carrying strong loss-of-function mutations in either egl-9 or vhl-1. We conclude that EGL-9 inhibits HIF-1 activity, as well as HIF-1 stability. Second, we identify the rhy-1 gene and show that it acts in a novel negative feedback loop to inhibit expression of HIF-1 target genes. rhy-1 encodes a multi-pass transmembrane protein. Although loss-of-function mutations in rhy-1 cause relatively modest increases in hif-1 mRNA and HIF-1 protein expression, some HIF-1 target genes are expressed at higher levels in rhy-1 mutants than in vhl-1 mutants. Animals lacking both vhl-1 and rhy-1 function have a more severe phenotype than either single mutant. Collectively, these data support models in which RHY-1 and EGL-9 function in VHL-1-independent pathway(s) to repress HIF-1 transcriptional activity.

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2α

“…Furthermore, an unknown protein (Y39A3CR.6, Supplementary data) with significant homology to mammalian HIF-1b-like protein was found (Ikeda and Nomura, 1997). Although the egl-9 mutant phenotype is HIF-1 dependent, the etiology is more complicated, since vhl-1-deficient worms have comparable HIF-1 levels, but are not egg-laying defective (Shen et al, 2006). This points to a TWIST1 is a target of HIF-2a EH Gort et al …”

“…Indeed, in addition to its role in regulating HIF protein stability through hydroxylation, the human EGL-9 homologue EGLN1 also suppresses HIF transcription in the nucleus . This may in part explain the higher transcriptional activity of HIF target genes and the more severe phenotype of egl-9-deficient animals (Shen et al, 2006). Furthermore, EGL-9 and the recently identified HIF-1 regulator RHY-1 function in a VHL-1-independent pathway to repress HIF-1 activity, which can explain a VHL-1-independent function of EGL-9 (Shen et al, 2006).…”

“…This may in part explain the higher transcriptional activity of HIF target genes and the more severe phenotype of egl-9-deficient animals (Shen et al, 2006). Furthermore, EGL-9 and the recently identified HIF-1 regulator RHY-1 function in a VHL-1-independent pathway to repress HIF-1 activity, which can explain a VHL-1-independent function of EGL-9 (Shen et al, 2006). To enrich for HIF-1-dependent genes from the primary candidates, we performed an additional selection using a hypoxia survival assay.…”

“…Quantitative PCR for C. elegans genes was performed using SYBR green (Applied Biosystems, Foster City, CA, USA). Specific primer sequences for F22B5.4 and inf-1 were kindly provided by Dr PowellCoffman (Shen et al, 2006). Q-PCR for human genes was performed using commercially available TaqMan assays for PGK1 (Hs99999906_m1), TWIST1 (Hs00361186), and hydroxymethylbilane synthase (HMBS; Hs00609297_m1; Applied Biosystems) using an ABI7900 analyzer (Applied Biosystems).…”

Feedback regulators of hypoxia-inducible factors and their role in cancer biology

“…Subsequently RHY-1 was identified as a negative feedback regulator of HIF function. 120 RNAi mediated inhibition of RHY-1, 121 increases HIF activity. The mechanism behind inhibition of HIF target gene expression by RHY-1 seems to be distinct from negative regulation by VHL.…”