MO290: Inhibition of the KDM4C and JMJD3 Histone Demethylases Alleviates the Tubular Renal Damage Triggered by Endoplasmic Reticulum Stress
Authors: Diaz-Ulnes Paula , Laura Saiz Alvarez Maria , Corte Viviana , Bernardo Florez Aida , Ruiz Bernet Cristian , Lopez-Larrea Carlos , Suarez-Alvarez Beatriz 
Published: May 2022

Abstract

BACKGROUND AND AIMS
Alterations in protein homeostasis in tubular cells lead to endoplasmic reticulum (ER) stress activating the unfolded protein response (UPR) pathway, which contributes to repair or aggravate the renal damage [1]. This pathway is initiated by three major protein sensors (IRE1α, PERK and ATF6) that activate their corresponding transcription factors (TF), XBP1, ATF4 and ATF6, respectively, to ultimately regulate the transcription of numerous genes essential for cell survival. However, an exacerbated activation of the UPR pathway can also lead to the expression of genes related to inflammation and fibrosis, cell death or autophagy contributing to perpetuate the renal damage [2]. The balance between these two processes (adaptive/maladaptive response) is mediated not only by the length and strength of the initial stimulus, but also by changes in the chromatin structure that may induce or repress gene transcription. Epigenetic changes are mainly mediated by the expression and recruitment of epigenetic enzymes, such as histone methyltransferases (HMTs) and demethylases (HDMs) to target genes in order to modify their expression. Thus, the aim of this study pursues to identify the epigenetic changes mediated by the histone methylation (H3K9 and H3K27) in the UPR pathway and to explore the role of the epigenetic drugs as potential treatments for kidney disease.

METHOD
The tubular epithelial cell line, HK2, was used to analyse the epigenetic changes in vitro before and after induction of ER stress mediated by Thapsigargin (Tg), an ER Ca2+ 2'-ATPase inhibitor and strongUPR inductor. Specific pharmacological inhibitors of the G9a and EZH2 HMTs, BIX-01 294 and GSK126, respectively, and of the JMJD3 and KDM4C HDMs, GSKJ4 and SD-70, respectively, or small interfering RNAs were used. The recruitment of these epigenetic enzymes and the presence of the H3K9me3 and H3K27me3 repressive histone marks were analysed by chromatin immunoprecipitation (ChIP) and coimmunoprecipitation assays.

RESULTS
HK-2 treatment with the BIX-01 294 or GSK126 pharmacological inhibitors or specific gene silencing of the G9a and EZH2 enzymes reveals an increase of the expression of ATF4 and XBP1 TFs, without inducing changes in ATF6 transcription. Moreover, this effect is additive to the one observed with Tg, indicating that changes in the chromatin structure are required for a full transcription and UPR activation. These results correlated with a lower recruitment of G9a and EZH2, and decreased H3K9me3 and H3K27me3 levels at the promoter region of ATF4 and XBP1 genes, corresponding with the increased transcription of these TFs. G9a and EZH2 HMTs act in coordination, so inhibition of G9a significantly reduces the recruitment of EZH2 and H3K27me3 levels to the regulatory region of ATF4 and XBP1 genes, and vice versa with EZH2 inhibition. In addition, enrichment in the global acetylation levels at histone H3 and H4 was observed, cooperatively facilitating the opening of the chromatin and the accessibility to transcriptional regulators. In accordance with these results, we demonstrate that blockage of the JMJD3 and KDM4C enzymes, responsible for demethylation of H3K9me3 and H3K27me3 marks, respectively, using the SD-70 and GSKJ4 epigenetic drugs, inhibits ATF4 and XBP1 expression under ER stress conditions and, consequently, the triggering of a maladaptative response.

CONCLUSION
Changes in the chromatin dynamics mediated by the H3K9me3 and H3K27me3 histone marks are key to regulate the expression of the UPR transcription factors ATF4 and XBP1 after ER stress activation. Pharmacological treatment with the epigenetic drugs SD-70 and GSKJ4 blocks the expression of these TFs and thus, the activation of the pathophysiological processes that contribute to aggravate renal damage triggered by the UPR pathway activation.
[1] Yan M., et al. Endoplasmic reticulum stress in ischemic and nephrotoxic acute kidney injury. Ann Med. 2018, 50: 381–390.
[2] Hetz, C.; et al. Mechanisms, regulation and functions of the unfolded protein response. Nat Rev Mol Cell Biol. 2020, 21: 421–438.