Degree Type

Dissertation

Date of Award

2010

Degree Name

Doctor of Philosophy

Department

Genetics, Development and Cell Biology

First Advisor

Jo A. Powell-coffman

Abstract

Oxygen is required for most organisms to survive, and plays a critical role in the evolution of life. However, oxygen deprivation (hypoxia) may occur in pathological conditions such as in solid tumors or normal physiological conditions such as during early embryonic development. When hypoxia occurs, organisms or cells will often respond with a series of changes to survive. One of the major responses is the changes in gene expression. Hypoxia inducible factors (HIF) regulate most of these gene expression changes.

The purpose of this study is to understand how HIF-1 transcriptional activity is regulated by prolyl hydroxylase protein EGL-9 and roles of EGL-9/HIF-1 in Pseudomonas aeruginosa. PAO1 resistance in Caenorhabditis elegans.

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 C. 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 abolish EGL-9-mediated repression of HIF-1 transcriptional activity. Further, analyses of new egl-9. alleles reveal that the evolutionarily conserved MYND zinc finger domain does not have a major role in regulating HIF-1 protein stability of transcriptional activity. 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.

Pseudomonas aeruginosa is a nearly ubiquitous human pathogen, and infections can be lethal to patients with impaired respiratory and immune systems. The P. aeruginosa PAO1 strain produces cyanide, and it can kill the nematode C. elegans within hours. Prior studies have established that strong loss-of-function mutations in the egl-9 gene protect C. elegans from P. aeruginosa PAO1 fast killing. To further understand the EGL-9 function in regulating HIF-1 transcription activity and its roles in P. aeruginosa resistance, we conducted a forward genetic screen. Here, we identify SWAN-1, an evolutionarily conserved WD-repeat protein, as an inhibitor of HIF-1-mediated gene expression. In genetic backgrounds that stabilize C. elegans HIF-1 protein, loss-of-function mutations in swan-1 increase the expression of hypoxia response genes and protect C. elegans from P. aeruginosa fast killing. Further, we show that EGL-9 forms a complex with SWAN-1, as assessed by yeast two hybrid or co-immunoprecipitation assays. These data support a model in which SWAN-1 and EGL-9 act in a common pathway to regulate HIF-1 transcriptional activity and modulate resistance to P. aeruginosa PAO1 fast killing

Copyright Owner

Zhiyong Shao

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

143 pages

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