Two distinct roles for EGL-9 in the regulation of HIF-1-mediated gene expression in Caenorhabditis elegans.
Oxygen is critically important to metazoan life, and the EGL-9/PHD enzymes are key regulators of hypoxia (low oxygen) response. When oxygen levels are high, the EGL-9/PHD proteins hydroxylate hypoxia-inducible factor (HIF) transcription factors. Once hydroxylated, HIFalpha subunits bind to von Hippel-Lindau (VHL) E3 ligases and are degraded. Prior genetic analyses in Caenorhabditis elegans had shown that EGL-9 also acted through a vhl-1-independent pathway to inhibit HIF-1 transcriptional activity. Here, we characterize this novel EGL-9 function. We employ an array of complementary methods to inhibit EGL-9 hydroxylase activity in vivo. These include hypoxia, hydroxylase inhibitors, mutation of the proline in HIF-1 that is normally modified by EGL-9, and mutation of the EGL-9 catalytic core. Remarkably, we find that each of these treatments or mutations eliminates oxygen-dependent degradation of HIF-1 protein, but none of them abolishes EGL-9-mediated repression of HIF-1 transcriptional activity. Further, analyses of new egl-9 alleles reveal that the evolutionarily conserved EGL-9 MYND zinc finger domain does not have a major role in HIF-1 regulation. We conclude that C. elegans EGL-9 is a bifunctional protein. In addition to its well-established role as the oxygen sensor that regulates HIF-1 protein levels, EGL-9 inhibits HIF-1 transcriptional activity via a pathway that has little or no requirement for hydroxylase activity or for the EGL-9 MYND domain.
Shao
Zhiyong
Z
Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011-3260, USA.
Zhang
Yi
Y
Powell-Coffman
Jo Anne
JA
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation / Q000187:drug effects; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D008954:Models, Biological; D009154:Mutation; D010100:Oxygen / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014170:Transformation, Genetic
D029742:Caenorhabditis elegans Proteins; D002614:Chelating Agents; C403074:Egl-9 protein, C elegans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D049452:Green Fluorescent Proteins; D010100:Oxygen