Originally published as Genetics Published Articles Ahead of Print on September 7, 2009.

Genetics, Vol. 183, 821-829, November 2009, Copyright © 2009
doi:10.1534/genetics.109.107284

Two Distinct Roles for EGL-9 in the Regulation of HIF-1-Mediated Gene Expression in Caenorhabditis elegans

Zhiyong Shao, Yi Zhang and Jo Anne Powell-Coffman1

Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011-3260

1 Corresponding author: 2108 Molecular Biology Bldg., Iowa State University, Ames, IA 50011-3260.
E-mail: japc{at}iastate.edu

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, HIF{alpha} 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.