GSK-J4 and suppression of the H3K27 demethylase UTX specifically activates expression of ATF4 and its target genes independently of PERK-eIF2α phosphorylation. GSK-J4 and suppression of the H3K27 demethylase UTX specifically activates expression of ATF4 and its target genes independently of PERK-eIF2α phosphorylation

UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase which is frequently mutated in various types of cancers. Although UTX appears to play a crucial role in cancer, its mechanisms remain poorly understood. Here we found that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces expression of the ATF4 (transcription activating factor 4) protein and its downstream target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3), and activates autophagy-related gene products (LC3A, LC3B and ATG5-ATG12). ATF4 induction by GSK-J4 did not entail either ATF6α processing or an increase of PERK and eIF2α serine 51 phosphorylation, indicating that this action is not triggered by canonical endoplasmic reticulum (ER) stress. No changes in expression levels of the endogenous ATF4 mRNA and exogenously expressed ATF4 proteins driven by the CMV promoter were detected, suggesting that the induction is not due to either transcriptional or post-translational regulation of ATF4. Although GSK-J4 efficiently induces apoptosis, it is unlikely that the induction is due to activation of the ATF4-CHOP axis. By using gene expression profiling experiments in conjunction with CRISPR editing and stable re-expression of UTX, we found that UTX specifically suppresses expression of ATF4 target genes. These findings highlight not only that GSK-J4, which has been considered an anti-cancer drug, is a strong inducer of ATF4, but also that UTX is an important component of this regulation and may contribute to oncogenesis via ATF4 regulation. Overall design: Stable wild type UTX expressing and empty vector control (CSII-CMV-IRES2-puro) clones were derived from two independent UTX CRISPR-Cas9 mutated HCT116 cell-lines CR1 and CR2. Total RNA was extracted from all 4 conditions in biological triplicates and subjected to Illumina HumanHT-12 v4.0 microarray profiling for identification of UTX-regulated target genes.