Genome-wide mRNA profiling identifies X-box-binding protein 1 (XBP1) as an IRE1 and PUMA repressor

Magdalena Gebert,Sylwia Bartoszewska,Piotr Madanecki,Aleksandra Sobolewska,David K Crossman,James F Collawn,Rafal Bartoszewski,Michał Dąbrowski,Aleksandra Cabaj,Jarosław Króliczewski

doi:10.1007/s00018-021-03952-1

Abstract

Accumulation of misfolded proteins in ER activates the unfolded protein response (UPR), a multifunctional signaling pathway that is important for cell survival. The UPR is regulated by three ER transmembrane sensors, one of which is inositol-requiring protein 1 (IRE1). IRE1 activates a transcription factor, X-box-binding protein 1 (XBP1), by removing a 26-base intron from XBP1 mRNA that generates spliced XBP1 mRNA (XBP1s). To search for XBP1 transcriptional targets, we utilized an XBP1s-inducible human cell line to limit XBP1 expression in a controlled manner. We also verified the identified XBP1-dependent genes with specific silencing of this transcription factor during pharmacological ER stress induction with both an N-linked glycosylation inhibitor (tunicamycin) and a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) (thapsigargin). We then compared those results to the XBP1s-induced cell line without pharmacological ER stress induction. Using next‐generation sequencing followed by bioinformatic analysis of XBP1-binding motifs, we defined an XBP1 regulatory network and identified XBP1 as a repressor of PUMA (a proapoptotic gene) and IRE1 mRNA expression during the UPR. Our results indicate impairing IRE1 activity during ER stress conditions accelerates cell death in ER-stressed cells, whereas elevating XBP1 expression during ER stress using an inducible cell line correlated with a clear prosurvival effect and reduced PUMA protein expression. Although further studies will be required to test the underlying molecular mechanisms involved in the relationship between these genes with XBP1, these studies identify a novel repressive role of XBP1 during the UPR.

Highlights

Endoplasmic reticulum (ER) stress can disrupt the folding and maturation of the secretory and membrane proteins and lead to the accumulation of unfolded proteins in the ER lumen, interruption of lipid synthesis, and deregulation of cellular calcium levels [1, 2]
Genes involved in the inflammatory responses [59], as well as genes not related to unfolded protein response (UPR) pathways including adipocyte and myogenic differentiation (C/EBP and MIST1) have been proposed as tissue-dependent X-box-binding protein 1 (XBP1) transcriptional targets [82]
The XBP1 has been widely accepted as an adaptive component of UPR that is responsible for facilitating protein folding and ER expansion

Summary

Introduction

Endoplasmic reticulum (ER) stress can disrupt the folding and maturation of the secretory and membrane proteins and lead to the accumulation of unfolded proteins in the ER lumen, interruption of lipid synthesis, and deregulation of cellular calcium levels [1, 2]. The buildup of misfolded proteins in ER leads to the activation of the unfolded protein response (UPR), a multifunctional signaling pathway that either promotes cell recovery [3], or initiates cell death if the ER stress remains unmitigated [4]. The UPR signaling pathways are initiated by three ER transmembrane sensors: inositol-requiring protein 1 (IRE1), protein kinase RNAlike ER kinase (PERK) and activating transcription factor 6 (ATF6). IRE1 removes a 26-base intron from X-box-binding protein 1 (XBP1) mRNA in an unconventional splicing reaction that results in a translational frameshift that leads to the production of a functional and highly active spliced XBP1 (XBP1) transcription factor [5,6,7,8,9]. The ER protein load is reduced by PERK-mediated phosphorylation of eIF2α which inhibits

Methods

Results

Conclusion