Two Distinct Roles for EGL-9 in the Regulation of HIF-1-Mediated Gene Expression in Caenorhabditis elegans |
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| Authors: | Zhiyong Shao Yi Zhang Jo Anne Powell-Coffman |
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| Affiliation: | Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011-3260 |
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| Abstract: | 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, HIFα 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.CELLS and tissues are often deprived of oxygen during normal development and during disease. Examples include animals that encounter hypoxic soil or aqueous microenvironments, mammalian tissues that receive insufficient oxygen when the cardiovascular system is taxed or disabled, and cells at the center of a poorly vascularized tumor. Most metazoans rely on aerobic respiration as a primary source of energy, and adaptation to hypoxia is of central importance. The hypoxia-inducible factor (HIF) transcription complexes have been termed master regulators of hypoxia response, because they regulate most hypoxia-induced changes in gene expression in animals as diverse as humans and the nematode Caenorhabditis elegans (Kaelin and Ratcliffe 2008). In mammals, these HIF targets include genes that regulate growth, energy metabolism, cellular differentiation, apoptosis, inflammation, and angiogenesis (Siddiq et al. 2007; Rankin and Giaccia 2008; Weidemann and Johnson 2008).The EGL-9/PHD proteins act as cellular oxygen sensors, and they are at the core of HIF regulatory networks. When oxygen levels are sufficiently high, PHD/EGL-9 proteins hydroxylate conserved proline residues in the HIFα subunits. Once hydroxylated, HIFα proteins bind to the von Hippel-Lindau tumor suppressor protein (VHL) (Bruick and McKnight 2001; Ivan et al. 2001; Jaakkola et al. 2001; Min et al. 2002). VHL targets HIFα for polyubiquitination and proteasomal degradation (Maxwell et al. 1999; Ohh et al. 2000).The nematode C. elegans has provided important insights into hypoxia signaling. The egl-9 gene was first identified in genetic screens for mutations that disrupted egg laying (Trent et al. 1983) and for mutations that conferred resistance to the bacterial pathogen Pseudomonas aeruginosa (Darby et al. 1999). Subsequent studies identified C. elegans EGL-9 as the oxygen-sensitive enzyme that controlled oxygen-dependent degradation of HIF-1, and EGL-9 was shown to be orthologous to mammalian PHD1, PHD2, and PHD3 (Epstein et al. 2001). C. elegans that carry a deletion in hif-1 are not able to survive development in hypoxia (Jiang et al. 2001; Padilla et al. 2002). hif-1 and egl-9 have been shown to have roles in other important processes, including heat acclimation, neural development, behavioral responses to oxygen or carbon dioxide, cyanide resistance, and aging (Gallagher and Manoil 2001; Jiang et al. 2001; Treinin et al. 2003; Bretscher et al. 2008; Chang and Bargmann 2008; Pocock and Hobert 2008; Chen et al. 2009; Mehta et al. 2009; Miller and Roth 2009; Zhang et al. 2009).Genetic analyses in C. elegans have shown that EGL-9 regulates HIF-1 via two distinct pathways: oxygen-dependent degradation of HIF-1 and an uncharacterized vhl-1-independent pathway in which EGL-9 represses HIF-1 transcriptional activity (illustrated in Figure 1A). In previous studies, we had discovered that the mRNA transcripts for HIF-1 target genes were expressed at much higher levels in egl-9 mutants, compared to vhl-1 mutants (Shen et al. 2006). Other studies had suggested that mammalian PHD proteins might also regulate HIF activity in some VHL-independent contexts (Ozer et al. 2005; To and Huang 2005). These findings supported the intriguing hypothesis that EGL-9/PHD proteins had VHL-independent roles that might not involve HIF hydroxylation.Open in a separate windowEGL-9 functions and models tested in this study. (A) EGL-9 regulates HIF-1 by two pathways, and they are illustrated here. First, EGL-9 controls oxygen-dependent degradation of HIF-1 (labeled pathway 1). EGL-9 hydroxlates HIF-1 on a conserved proline residue (P621), and this enables binding of HIF-1 to the VHL-1 E3 ligase. HIF-1 is then degraded. Molecular oxygen, Fe(II), and 2-oxoglutarate are required for the hydroxylation reaction. EGL-9 also suppresses expression of HIF-1 targets by a second pathway that does not require VHL-1 (labeled pathway 2 here). (B) Initial alternative models for the VHL-1-independent functions of EGL-9 (pathway 2). Each model predicts a different combination of experimental outcomes. Model a postulates that pathway 2 (like pathway 1) requires hydroxylation of HIF-1 proline 621. Model b is that EGL-9 hydroxylates a different target to inhibit HIF-1 transcriptional activity. This model predicts that all EGL-9 functions would be abrogated by mutations or treatments that eliminated EGL-9 hydroxylase activity. Model c is that EGL-9 represses HIF-1-mediated transcription by a mechanism that does not require EGL-9 hydroxylase activity.In this study, we investigate the vhl-1-independent mechanism by which C. elegans EGL-9 represses HIF-1 activity. We find that while hydroxylation of HIF-1 at proline residue 621 by EGL-9 is required for HIF-1 destabilization, it is not essential for the vhl-1-independent functions of EGL-9. Further, we show that the two EGL-9 pathways have differing sensitivities to mutations or pharmacological treatments that impair hydroxylase activity. Collectively, these data show that EGL-9 represses HIF-1 transcriptional activity via a pathway that has little or no requirement for EGL-9 hydroxylase activity. |
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