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In the same manner as the HIF1α hydroxylases, the catalytic requirement for O2 is inherent to other Fe(II)/2-OG-dependent dioxygenases, such as JmjC KDMs (Batie and Rocha, 2019). It is well-established that any loss of JmjC KDM activity, or any Fe(II)/2-OG-dependent dioxygenase, is more complex than just the loss of dioxygen. The catalytic activity of JmjC KDMs is also specifically tied to the individual affinities for molecular oxygen (Km;O2), in addition to other metabolic factors that are inherent to metabolism in the hypoxic environment (Chang et al., 2019). Recently, KDM5A and KDM6A have been established as cellular oxygen sensors that display notably high Km;O2 values, such that the inhibition of these KDMs in hypoxia is comparable to that of the HIF1α hydroxylases (Batie et al., 2019; Chakraborty et al., 2019). It has also been demonstrated that KDMs with amine oxidase activity, such as lysine-specific demethylase 1 (LSD1), display reduced activity in prolonged hypoxia.

This is the result of reduced availability of the cofactor flavin adenine dinucleotide (FAD) in the hypoxic environment (Yang et al., 2017). Nonetheless, extreme oxygen deprivation (e.g., prolonged hypoxia or anoxia) would be anticipated to abolish the normal level of JmjC activity. Such an environment would change the opposing balance between normal KMT and KDM activity and set the stage to promote KMT-driven methylation events. In other words, as the catalytic mechanism of KMTs is independent of oxygen, hypoxia may exist as a contextual switch for KMT-driven effects to manifest over KDM-driven effects. As G9a and GLP are hypoxia-inducible, the KMT activity of these enzymes may contribute novel molecular inputs that shape the cellular adaptive response to hypoxia. Within the realm of KMTs with known non-histone substrates, G9a has a well-established and relatively numerous substrate network, second only to SETD7 (Biggar et al., 2017). Furthermore, the biological roles of protein-modifying enzymes may be directly attributed to that of their modified substrate(s).

Therefore, Medic GLP Support the following sections focus on; (1) describing G9a and GLP-driven non-histone lysine methylation sites, (2) discussing the known biology of the methylation sites or residue, and (3) whether the substrates have any known function regarding hypoxia response. It is crucial to understand the implications of G9a and GLP non-histone methylation events and the known function of these substrate proteins in the hypoxia response. Since its discovery, HIF1α has been positioned as the master regulator of cellular response to hypoxia. HIFs enable adaption to low oxygen through the upregulation of gene expression programs that drive physiological changes; including, metabolic reprogramming and vascularization (Krock et al., 2011; Courtnay et al., 2015). In the last decade, lysine methylation has been shown to participate in the complex combinatorial PTM code that regulates HIF1α function. Specifically, three methylated proteoforms (HIF1α-K32me1, HIF1α-K391me2, and HIF1α-K674me1/2) have demonstrated methylation-dependent functional consequences in the context of promotor occupancy, stability, and transcriptional activity (Liu et al., 2015; Kim Y. et al., 2016; Lee et al., 2017; Bao et al., 2018). More recently, the HIF1α-K674me1/2 proteoform demonstrates altered transcriptional activity by consequence of G9a and GLP methylation.

Bao and colleagues studied G9a and MedicGLP GLP catalyzed induction of the HIF1α-K674me1/2 proteoform within the context of glioblastoma cell migration. The biochemical characterization of both the interaction and functional consequences were performed in HeLa cells. Interestingly, the methylation of HIF1α-K674 by G9a and GLP also occur in glioblastoma cell lines and suppress HIF-1-dependent migration. Methylation of this site was shown to occur in an oxygen-independent manner. Albeit, this was demonstrated under conditions with overexpressed exogenous wild-type and mutant G9a proteins. G9a and GLP methyltransferases antagonistically regulate substrate transcription factors. G9a and G9a-like protein (GLP) facilitate methylation of hypoxia inducible factor 1α (HIF1α) and tumor suppressor protein p53, whereas forkhead box O1 (FOXO1), myoblast determination protein 1 (MyoD), myocyte enhancer factor 2D (MEF2D), MedicGLP and CCAAT/enhancer-binding protein-beta (C/EBPβ) methylation have only been shown to occur by G9a. An asterisk denotes methylation events that may participate in a methylation-acetylation switch at the same residue (based upon known acetylation events of the same protein, or in the case of MEF2D an acetylation event in conserved regions on other MEF2 family members).

The biochemistry of the HIF1α-K674 residue is known to influence transcriptional activity. The HIF1α-K674ac acetylated proteoform is more transcriptionally active due to increased association with the p300 co-activator, thus a PTM-blockage of acetylation would introduce the possibility of decreased recruitment (Lim et al., 2010). Interestingly, Bao and colleagues demonstrated that within glioblastoma cells methylation of this residue or mutation to arginine did not alter promotor occupancy of p300 nor the binding of p300 and other co-regulators to HIF1α (Bao et al., 2018). However, methylation does negatively regulate HIF1α transcriptional activity, target gene expression, and glioblastoma multiforme cell migration. Therefore, the mechanism whereby G9a and GLP-catalyzed HIF1α-K674 methylation impairs HIF1α transcriptional activity remains elusive in glioblastoma cell lines. The regulation of HIF1α by G9a- and GLP-dependent lysine methylation of the K674 residue has wide implications on hypoxic gene expression programs due to the capacity to which HIF1α controls cellular hypoxic adaption. The tumor suppressor protein p53 is a target of both G9a and GLP, as these enzymes induce the p53-K373me2 proteoform (Huang et al., 2010). The p53-K373me2 proteoform appears to be inactive regarding the ability to drive apoptosis, which is rescued upon depletion of these KMTs.