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

Hypoxia tolerance is mainly controlled by the hypoxia signaling pathway and HIF-1α/2α 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-1α/2α. MYLIP interacts with HIF-1α/2α and catalyzes K27-linked polyubiquitination at lysine 118/442 (HIF-1α) or lysine 117 (HIF-2α). This modification induces proteasomal degradation of HIF-1α, 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 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.