Genome-wide analyses of expression data and HIF-1 binding sites provide insights to the HIF-1 hypoxia-inducible factor in Caenorhabditis elegans

摘要

ABSTRACTThe hypoxia-inducible factor (HIF) has been described as the master regulator of transcriptional changes that enable hypoxia adaptation. HIF is conserved and expressed by all extant metazoan species analyzed to date (LOENARZ et al. 2011; SEMENZA 2000; SEMENZA 2012). In this dissertation, I used C. elegans as a model system to study HIF-1 function and regulation. In the first part of my thesis, we studied the cross talk between HIF-1 and SKN-1. We found that SKN-1 transcriptionally regulates egl-9 to inhibit HIF-1 protein levels and activity. We propose that this interaction enables animals to adapt quickly to changes in cellular oxygenation and to better survive accompanying oxidative stress.The second part of the thesis is dedicated to understand the phenotypes of 4 known HIF 1 negative regulator mutants and the consequences of HIF-1 over-activation. Affymetrix microarrays were used to measure the whole genome mRNA expression in vhl-1(ok161), rhy 1(ok1402), egl-9(sa307), and swan-1 (ok267); vhl-1(ok161) compared to N2 wild-type animals. In supporting the model that RHY-1, EGL-9 and SWAN-1 act in common pathway(s) to repress HIF-1, our data show that the mutant phenotypes of rhy-1, egl-9 and swan-1 loss-of-function are similar at the molecular level. Prior studies suggest that besides hypoxia resistance, misregulation of HIF-1 disrupts multiple processes, including pathogen resistance and brood size. In consistent with this, we found that genes up-regulated by HIF-1 over-activation converge with genes involved in immune response pathways; and genes commonly misregulated in HIF-1 over-activation mutants are enriched in cell-cycle and reproduction functions. Intriguingly, we found that HIF-1 and DAF-16 converge on key stress-responsive genes and function synergistically to protect C. elegans from moderate hypoxia stress.The third part of the thesis combines microarray analyses and ChIP-seq to understand HIF-1 function. The microarray study in this part focuses on HIF-1-dependent gene expression changes under short-term hypoxia (2 hours, 0.5% oxygen). ChIP-seq is aimed to identify HIF-1 direct targets. We found that HIF-1 mediated hypoxia gene expression changes are involved in multiple biological processes, genes involved in lip metabolism are newly identified by this study. Collectively, these functions are important for C. elegans development and survival under hypoxia. We also found that short-term hypoxia-responsive genes overlap with H2S responsive genes, and HIF-1-dependent hypoxia-responsive genes overlap with genes regulated by DAF-16. Further, we found that potential HIF-1 direct targets identified by ChIP-seq are enriched in the differentially expressed gene lists identified by microarray in response to hypoxia treatment and constitutive HIF-1 high activation.