MYLIP attenuates hypoxia tolerance by inducing K27-linked polyubiquitination and subsequent proteasomal degradation of HIF-a

Hypoxia tolerance is mainly controlled by the hypoxia signaling pathway and HIF-1a/2a serve as master regulators in this pathway. Here we identify MYLIP, an E3 ubiquitin ligase thought to specifically target lipoprotein receptors, as a negative regulator of HIF-1a/2a. MYLIP interacts with HIF-1a/2a and catalyzes K27-linked polyubiquitination at lysine 118/442 (HIF-1a) or lysine 117 (HIF-2a). This modification induces proteasomal degradation of HIF-1a, resulting in inhibition of hypoxia signaling. Furthermore, Mylip-deficient bluntsnout bream, zebrafish and mice are more tolerant to hypoxia. These findings reveal a role for MYLIP in regulating hypoxia signaling and identify a target for developing fish strains with high hypoxia tolerance for the benefit of the Overall design: M. amblycephala (2 mpf) of similar weight were selected for hypoxia experiments. The flask was filled with 200 ml of water. Three M. amblycephala were placed in a flask under normoxia (21% O2) or hypoxia (5% O2) for 2 hours. These above sampled fish were euthanized by a concentration of 0.3 mg/L MS-222, and the brain was dissected and immediately stored in liquid nitrogen for total RNA extraction. Experiments were repeated five times (n = 5 per group). Clustering of indexed samples was performed on a cBot cluster generation system using the TruSeq PE Cluster Kit v4-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 4000 platform and paired-end 150bp reads were generated. Differential expression analysis of two conditions/groups was performed using the DESeq R package (1.10.1). DESeq provides statistical routines to determine differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg's approach to control for the false discovery rate. Genes with an adjusted P value < 0.05 found by DESeq were considered to be differentially expressed.