Upregulation of pro-apoptotic genes by the accumulation of unfolded proteins

The unfolded protein response (UPR) is a complex cellular reaction to a buildup of excess unfolded and/or misfolded proteins in the endoplasmic reticulum. One branch of the UPR is the PERK-pathway, which occurs when the chaperone BiP associates with unfolded proteins which leads to the activation of PERK. PERK then phosphorylates eIF2ɑ, leading to inhibition of global protein synthesis. The focus of our project last year were the genes GADD34 and CReP. BiP, GADD34, and CReP gene expression were measured using in-situ hybridization in unstressed embryos and embryos treated with thapsigargin to induce ER stress. The difference in the expression of BiP during unstressed and stressed conditions was used as a positive control for our experiment. First, the embryos were collected, and then 24 hpf embryos were put through either no treatment or thapsigargin treatment for either 4 or 24 hours, fixed with paraformaldehyde, and analyzed by in-situ hybridization. A large increase in expression of BiP, our positive control, was observed throughout the embryos upon stress induction. GADD34 expression also increased following stress, primarily in the head of the embryos. CReP showed increased gene expression mainly in the tails of embryos treated for 4 hours and in the heads of embryos treated for 24 hours. In addition, GADD34 and CReP may have protective effects against apoptosis, as is seen when cell death is reduced when GADD34 is knocked out. As such, we now plan to study the effects on the main apoptotic signals during ER stress, which are p63, PUMA, and CHOP. The in-situ hybridizations for the activity of these genes, probes must be designed, which was the main goal of our project this year. Designing the probes involved a series of genetics lab techniques, including primer design, polymerase chain reaction, DNA cloning, bacterial transformation, midi-prepping, and restriction digestion.