Reactive oxygen species (ROS) encompass a group of molecular oxidants generated as by-products of metabolic processes or by exogenous agents [[1], [2], [3]]. Intracellular ROS function as specific signaling molecules for the host defense mechanisms and proliferative responses and can trigger chemical reactions that lead to oxidative damage to DNA and other cellular components [[4], [5], [6]]. Because of the dual nature of ROS, an imbalance between ROS generation and removal has been linked to various pathophysiological conditions such as aging, cancer, cardiovascular disease, and neurodegenerative disorders [[7], [8], [9], [10], [11]]. Cells have evolved sensing and signaling mechanisms for counteracting oxidative stress to maintain physiological homeostasis, which include enzymatic and non-enzymatic antioxidant and detoxification responses against elevated intracellular ROS [3,5].

NRF2 (NF-E2 Related Factor 2), also known as NFE2L2 (NFE2 like bZIP transcription factor 2), is a transcription factor that plays crucial roles in protecting cells from oxidative and xenobiotic stress by orchestrating the expression of cytoprotective genes [12,13]. The molecular mechanisms for regulating NRF2 activity at the posttranslational stage have been well described [14,15]. Under homeostatic condition, NRF2 is negatively regulated by KEAP1 (kelch-like ECH-associated protein 1), which promotes the ubiquitin-dependent degradation of NRF2. Upon oxidative stress, NRF2 is released from KEAP1, translocated into the nucleus, and binds to antioxidant response elements (AREs) and electrophile response elements (EpREs) to activate an array of antioxidant and detoxification genes [[16], [17], [18]]. Functions of NRF2 are also modulated by posttranslational modifications, such as phosphorylation and acetylation, which affect protein stability, nuclear translocation, and binding to target sites [13,19]. In addition, several factors have been implicated in regulating NRF2 expression, which include sequence-specific DNA binding transcription factors, such as AhR, NFkB, p53, glucocorticoid receptor, RXRA, and NRF2 itself, and epigenetic regulators involved in DNA/histone modifications [[20], [21], [22], [23], [24], [25], [26]].

Lysine methylation of histones is a posttranslational modification that affects the chromatin structure and function depending on the site and status of modification [27]. Trimethylated histone H3K9 (H3K9me3) or trimethylated histone H3K27 (H3K27me3) at the promoter region is correlated with inhibited transcription, whereas increased occupancy of trimethylated histone H3K4 (H3K4me3) at the promoter is a hallmark of gene activation [28,29]. Histone lysine methyltransferases (KMTs) are enzymes that catalyze the transfer of methyl groups to lysine and have been implicated in the regulation of NRF2 [26,30]. The EZH2 histone methyltransferase, the catalytic subunit of Polycomb repressive complex 2 (PRC2), targets the NRF2 promoter, leading to the enrichment of H3K27me3 and inhibition of NRF2 expression in lung cancer cells [31]. Mixed-lineage leukemia protein (MLL) is associated with increased H3K4me3 at the NRF2 promoter in chemo-resistant colon cancer cells [32]. In addition, it has recently been reported that ROS-induced oxidative stress by cerium oxide nanoparticles (CeO2 NPs) results in increased MLL1 (KMT2A) binding and enrichment of H3K4me3 at the NRF2 promoter, leading to the transcriptional activation of NRF2 in human keratinocytes HaCaT cells [33]. On the other hand, roles of histone demethylases (KDMs), enzymes that functionally counteract KMTs, in NRF2 expression are not fully understood. Interestingly, CeO2 NPs-induced transcriptional activation of NRF2 is accompanied with reduced H3K27me3 at the NRF2 promoter, which is likely mediated by KDM6 family of histone demethylases, such as UTX (KDM6A) and JMJD3 (KDM6B) [33,34].

In this study, we hypothesized that epigenetic factors involved in dynamic changes in H3K4me3/H3K27me3 histone modifications at the NRF2 promoter play important roles in antioxidative response and investigated the roles of MLL1 histone lysine methyltransferase and UTX histone demethylase in ROS-induced transcriptional activation of NRF2. We chose MLL1 and UTX because MLL1, in addition to the increased binding at the NRF2 promoter upon CeO2 NPs treatment, has been implicated in the signal-induced activation of genes containing H3K4me3/H3K27me3 or H3K4me3/H3K9me3 bivalent promoter [[35], [36], [37]], and UTX is a major histone demethylase targeting H3K27me3 [34]. Decreased cell viability and failed activation of NRF2 in cells expressing shRNAs targeting KMT2A (for MLL1) or KDM6A (for UTX) upon oxidative stress suggested the crucial roles of these epigenetic modifiers in response to ROS-generating stress. In addition, ChIP analysis demonstrated the selective binding of MLL1 and UTX to the NRF2 promoter. Moreover, the exogenous expression of FLAG-NRF2 in MLL1-or UTX-depleted cells led to significant recovery in the antioxidative responses. Moreover, ChIP-seq and RNA-seq analyses provided a framework for the MLL1/UTX-dependent regulation of antioxidant and detoxification systems. These findings highlight the important roles of MLL1/UTX in redox homeostasis and may provide potential therapeutic targets for pathophysiological conditions associated with the cellular responses to oxidative stress.