Hypoxia (O2 deprivation) plays a central role in diverse human diseases, including ischemic stroke, myocardial
infarction, pulmonary hypertension, Cerebral Palsy, COVID-19, and cancer. Metazoans respond to hypoxia by
employing the conserved hypoxia response pathway. The pathway senses O2 through a prolyl hydroxylase
(PHD) enzyme, which uses O2 to hydroxylate specific proline side chains on the Hypoxia Inducible Factor a
(HIFa). Once hydroxylated, HIFa is ubiquitinated by the Von Hippel-Lindau (VHL) ubiquitin ligase, resulting in
its proteolysis. When O2 is abundant, HIFa is unstable. When hypoxia ensues, PHD enzymes lack O2 to
hydroxylate HIFa, resulting in HIFa stabilization and the transcriptional regulation of multiple target genes that
help the organism survive. Under some circumstances (e.g., solid tumors, stem cell niches), HIFa is activated
despite adequate O2 levels (i.e., the Warburg effect), but how the response differs under aerobic conditions is
unclear. While the HIFa pathway has been well studied in tissue culture, a full understanding of how it
operates in specific tissues (particularly neurons) in vivo to provide tailored responses is needed.
This proposal takes advantage of genetics and an intact, isogenic model organism (C. elegans) that
can thrive under hypoxia, and whose environment and genetics can be controlled with fidelity and
reproducibility. C. elegans possess single genes for the PHD (EGL-9), the VHL (VHL-1), and the HIFa (HIF-1).
The overall premise of this proposal is that the hypoxia response pathway pathway protects against hypoxic
damage by (1) removing mitochondria through mitophagy, which eliminates a source of ROS, and by (2)
mobilizing antioxidant metabolism, which detoxifies ROS during hypoxia and reoxygenation. A better
understanding of the pathway response will provide therapeutic targets for diseases associated with hypoxia.
Preliminary ChIP-seq, RNA-seq, and metabolomics suggest that HIF-1 promotes gluconeogenesis, the
pentose phosphate pathway, and antioxidant generation. We hypothesize that HIF-1 promotes this metabolic
reprograming by binding an enhancer sequence and activating the expression of the PEP carboxykinase pck-
1, a key enzyme for moving metabolites through gluconeogenesis. Aim 1 tests this hypothesis by using
CRISPR/Cas9 editing to remove this enhancer, then testing for the effects on HIF-1 binding, pck-1 and global
gene expression, metabolism, oxidative stress resistance, neurodegeneration, and hypoxia survival.
Preliminary cell biological approaches with a genetically encoded fluorescent reporter for mitophagy
suggest that HIF-1 promotes mitophagy. We hypothesize that HIF-1 promotes mitophagy by binding
enhancer sequences and activating the expression of the mitophagy receptors fndc-1 and dct-1. Aim 2 tests
this hypothesis by using CRISPR/Cas9 editing to remove these enhancers, then testing for the effects on HIF-
1 binding, global gene expression, mitophagy and bulk autophagy, metabolism, oxidative stress resistance,
neurodegeneration, and hypoxia survival.