HIF-1-independent Hypoxia Response of hsp-16.1 in Caenorhabditis elegans

Abstract

During normal development and disease state, cells and organisms encounter low oxygen environments, a condition that is termed hypoxia. To overcome cellular damage and death that may result from oxygen deprivation, cells respond by expressing numerous hypoxia response genes, many of which are controlled by the hypoxia-inducible factor-1 (HIF-1). Recently, the cellular responses to hypoxia in the HIF-1-indedepent manner have been reported, but little is known about their molecular mechanisms. The nematode Caenorhabditis elegans (C. elegans) is one of the most important model systems for studying hypoxia response. In this study, I have identified core regulators that mediate hypoxia response and have examined how the expression of a hypoxia responsive gene can be modulated in the HIF-1-independent manner in C. elegans. To better understand the mechanisms of HIF-1-independent hypoxia response, I have characterized the molecular basis of the hypoxia response of the hsp-16.1 gene in C. elegans. This gene has been shown to be induced under hypoxic conditions in hif-1 mutant animals, which demonstrates that hypoxia response of hsp-16.1 is independent of hif-1. The HIF-1-independent hypoxia response of hsp-16.1 requires a conserved DNA sequence (CAC(A/T)CT) at its promoter region. Using affinity purification followed by LC-MS/MS, HMG-1.2 has been identified as a protein that binds to the conserved region of hsp-16.1 under hypoxic conditions. To further characterize the functional role of HMG-1.2 in hypoxia response, I employed a method termed functional gene network. With this, I screened most of C. elegans orthologs shared with other species for biological interaction with hmg-1.2 such as gene expression, co-citation, protein-protein interaction, and genetic interaction. I identified candidate genes that might interact with hmg-1.2. To validate these predictions, I performed RNAi to disrupt genes which were predicted to interact with hmg-1.2. I identified the chromatin modifiers isw-1 and hda-1, histone H4, and the NURF-1 chromatin remodeling factor as new components of the hif-1-independent hypoxia response. These results suggest that the modulation of nucleosome positioning at the hsp-16.1 promoter may be important for the hif-1-independent hypoxia response mechanism. In addition, I found that calcium ions are necessary for the induction of hsp-16.1 in hypoxic conditions, and that calcineurin acts independently of hif-1 to modulate the cellular response to hypoxia. While the HIF-1 mediated gene regulation under hypoxic conditions has already been extensively studied, very little is known on HIF-1 independent response to hypoxia. Here I present a novel mechanism of hypoxia response where chromatin modification and calcium homeostasis may act independently of HIF-1 in C. elegans.