Systematic Identification of Gene Activities Promoting Hypoxic Death

Abstract

The sensitivity of an organism to hypoxic injury varies widely across species and among cell types. However, a systematic description of the determinants of metazoan hypoxic sensitivity is lacking. Toward this end, we screened a whole-genome RNAi library for genes that promote hypoxic sensitivity in Caenorhabditis elegans. RNAi knockdown of 198 genes conferred an invariant hypoxia-resistant phenotype (Hyp-r). Eighty-six per cent of these hyp genes had strong homologs in other organisms, 73 with human reciprocal orthologs. The hyp genes were distributed among multiple functional categories. Transcription factors, chromatin modifying enzymes, and intracellular signaling proteins were highly represented. RNAi knockdown of about half of the genes produced no apparent deleterious phenotypes. The hyp genes had significant overlap with previously identified life span extending genes. Testing of the RNAi's in a mutant background defective in somatic RNAi machinery showed that most genes function in somatic cells to control hypoxic sensitivity. DNA microarray analysis identified a subset of the hyp genes that may be hypoxia regulated. siRNA knockdown of human orthologs of the hyp genes conferred hypoxia resistance to transformed human cells for 40% of the genes tested, indicating extensive evolutionary conservation of the hypoxic regulatory activities. The results of the screen provide the first systematic picture of the genetic determinants of hypoxic sensitivity. The number and diversity of genes indicates a surprisingly nonredundant genetic network promoting hypoxic sensitivity.

Role of oxygen consumption in hypoxia protection by translation factor depletion

“…One-generation feeding RNAi was performed as described previously (Timmons, 2006b;Mabon et al, 2009). Briefly, two gravid adult worms were left on agar RNAi plates with 2-day-old RNAi bacterial lawns for 3h to obtain 30-50 eggs per plate.…”

“…In Caenorhabditis elegans (Maupas 1900), we have performed screens for genes whose mutant phenotype is improved organismal survival after hypoxia (Scott et al, 2002;Anderson et al, 2009;Mabon et al, 2009). Each of these screens has identified genes known to regulate protein homeostasis.…”

“…rars-1(gc47) animals have añ 50% reduction in global translation rate consistent with the essential role of aminoacyl-tRNA synthetases such as RARS-1 in protein translation. The discovery that RARS-1 along with other aminoacyl-tRNA synthetases (Anderson et al, 2009) and a few implicated translation factors (Mabon et al, 2009) strongly control hypoxic survival motivated a more systematic study of the effect of translation factor knockdown on hypoxic injury. In order to establish mammalian relevance and therapeutic potential, we first tested the ability of translation factor knockdown to protect cultured mouse hippocampal neurons.…”

Regulation of Anoxic Death in Caenorhabditis elegans by Mammalian Apoptosis Signal-Regulating Kinase (ASK) Family Proteins

“…Under anoxic conditions, nematodes decrease their metabolic rate and arrest their cell cycle progression (Padilla et al 2002). Over the last decade, several studies have examined the mutations that affect the survival rate under lethal anoxia (Scott et al 2002;Nystul et al 2003;Mendenhall et al 2006;Samokhvalov et al 2008;Anderson et al 2009;Mabon et al 2009;Menuz et al 2009), but the signaling pathways that regulate survival under anoxic conditions have not fully been elucidated.…”

Non‐apoptotic cell death in Caenorhabditis elegans

“…Hypoxic preconditioning is dependent on CED-4, while other molecules of the core apoptotic machinery (EGL-1, CED-3, and CED-9) are not required. Genome-wide RNAi screens have identified many genes conferring hypoxia sensitivity in the nematode (Mabon et al, 2009). These genes are grouped in diverse functional classes, encoding proteasomal components and molecules regulating transcription and translation, chromatin remodeling, and nucleosomal histone modifications, among others.…”