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Lysine demethylases, which are also known as KDM's, are enzymes that catalyze the methyl group removal on the histone lysine and arginine residues. Histone lysine methylation was identified as a permanent and irreversible chromatin marker process. However, it has now been described as a reversible process by two different demethylase subfamilies. These subfamilies are the flavin-dependent KDM1 subfamily and the Fe (II)‑ and 2OG‑dependent JmjC-domain-containing KDM2-8 subfamily. They have an effect on the gene expression, repair and replication of DNA, genome stability, and the regulation of the chromatin state at specific loci.

The role of demethylases in aging and the changes in the expression and activity of demethylases with aging are still being investigated. However, the effect of demethylases on retarding cellular senescence has been observed in recent studies.

KDMs

LSD1, which is a member of the flavin-dependent superfamily, was the first histone demethylase discovered. Following that, the first identified JmjC-containing histone demethylase was KDM2A. Other KDM’s are illustrated in the table "Demethylases".

Demethylases

KDM1 Family

The KDM1 family is formed by two amine oxidase superfamily members, KDM1A and KDM1B, that couple the oxidation of substrates to the flavin adenine dinucleotide (FAD) reduction. According to studies, this process contains electron transfer steps that require a pair of electrons on methyl lysine. As a result, Nε-trimethyl-lysyl residues are not accepted as substrates by KDM1s.

KDM1 homologs can be found in organisms varying from fission yeast to mammals, each having at least two genes related to KDM1. KDM1A, also known as LSD1 (lysine-specific demethylase 1), was first identified by Shi et. al (2004). This enzyme is an amino oxidase homolog that contains highly conserved flavin and removes the mono- and di-methylated lysine from lysine 4 or 9 of H3, which depends on the cellular condition. KDM1A includes a SWIRM domain discovered in chromatin-modifying proteins and oxidizes methyl groups leading to the generation of formaldehyde. According to studies, KDM1A demethylates at H3K4me1 and H3K4me2 resulting in transcriptional inactivation via the interaction between the tower domain of CoREST and KDM1A. Nevertheless, transcriptional activation occurs due to the complex of the androgen receptor and KDM1A, demethylating H3K9me1 and H3K9me2.

KDM1A also demethylates non-histone proteins such as p53 and regulates their stability, abundance, or activity. Hence, KDM1A can act as a transcriptional activator and block the function of p53 by demethylating it, which leads to the prevention of p53 and 53BP1 interaction. In response to DNA damage, KMD1A also demethylates the transcription factor E2F1. Then, stabilization of E2F1 and promotion of apoptosis occurs. Moreover, the activity of KMD1A indirectly affects the global DNA methylation levels as the central DNA methyltransferase stability is subjected to the cycle of lysine methylation and demethylation, which is regulated by KDM1A.

KDM1B is similar to KDM1A as it is also a FAD-dependent amino oxidase homolog that contains a SWIRM domain and targets H3K4me1 and H3K4me2. However, they are not completely identical as KDM1B cannot generate a complex with CoREST due to not having a tower domain. Although KDM1B's regulatory roles are still being investigated, it has been discussed that they have a function in the maternal imprinting in oocytes.

The JMJC Domain-Containing Demethylases

The Jumonji C (JmJC) domain-containing demethylases are the biggest class of histone demethylases, which contain almost 20 lysine-specific demethylases. They are different from KDM1 family due to their capability of removing trimethylations. From KDM2 to KDM8, the JmjC domain generates the catalytic core through utilizing its β-barrel secondary structure. The confirmations of β-barrel allow Fe(II) and 2OG to demethylate the methylated lysine. The JMJC domain-containing demethylases also include a JmjN domain on the N-terminal of the JmjC domain, which interacts with surrounding domains while supplying structural stability without generating part of the catalytic pocket.

The subfamilies of KDM2, KDM4, KDM5, and KDM7 each have PHD domains that identify the H3K4me3 epigenetic chemical alteration by acting as a domain that binds nucleosomes. A leucine-rich repeat and F-box domain in the KDM2 subfamily promotes protein-protein interactions, while a CXXC zinc finger domain differentiates unmethylated CpGs DNA. TUDOR domain, which has a role in binding the methylated histone molecules and RNA, is contained by the KDM4 subfamily. KDM5 contains ARID that functions as a DNA binder and modifies chromatin assembly. KDM6A has a tetratricopeptide repeat that forms a scaffold for protein-protein interactions, as well as a GATA-like zinc finger domain that has a role in binding the DNA sequence found in gene regulatory areas.

Aging

Effect of Aging on Demethylase Activity

Both endogenous factors, such as changed expression or activity of DNA methyltransferases and demethylases, and exogenous factors, such as diet, medications, and UV light, affect the age-dependent alterations in methylation. Aging decreases DNA demethylase activity while increasing DNA methyltransferase activity, resulting in the increased DNA methylation, which was observed in the Klotho gene in the aortas of elderly mice. The compound H reduced aging-related arterial stiffness and hypertension via activating DNA demethylase, which raised the expression of renal SKL and, as a result, the Sirt1-AMPK-eNOS pathway was activated.

Western blot analysis of Klotho expression in the (a) kidney (b) serum of aged mice. (c) mRNA expression of Klotho in kidneys.

Role of Demethylases in Aging

There are several studies conducted to investigate the effects of demethylases on aging. Positive impacts of demethylases on cellular aging via repression was discussed in various studies. Alvares et. al (2014) observed that activities of histone 3 lysine 4 (H3K4) demethylase enhanced the chromatin state maintenance during stressful growth environments, which led to an inhibition in the aging of post mitotic somatic cells and proliferative germ cells. In the study, H3K4 demethylases of Caenorhabditis elegans, RBR-2 and SPR-5, were discovered to improve the post mitotic longevity of stress-resistant daf-2 adults, change the methylated H3K4 pools, and promote the silencing of several daf-2 target genes. Consequently, H3K4 demethylases may have a conserved function in repressing cellular aging by altering chromatin regarding physiological stress.

H3K4 demethylases and somatic longevity.

Furthermore, Jiang et. al 2021 demonstrated that the N6-methyladenosine (m6A) demethylase FTO in granulosa cells (GCs) slow downed FOS-dependent ovarian aging. ROS accumulation during ovarian aging causes low expression of the demethylase FTO in GCs, and the resulting increase in m6A retards the FOS-mRNA degradation for upregulating FOS in GCs. Ultimately, the aging of GCs is promoted followed by the ovarian aging acceleration. Moreover, data linking m6A in GCs to ovarian aging leads to female reproductive failure as well as the development of procedures for aged ovary rejuvenation and the preservation of female fertility. Yet, FTO acts as a protein that retards senescence by m6A, as FOS knockdown significantly alleviates the aging of FTO-knockdown GCs. This indicates that FTO in GCs retards the FOS-dependent ovarian aging. Hence, they have the potential to be a diagnostic and therapeutic target for ovarian aging and age-related reproductive disorders.

The effect of FTO as an m6A demethylase in ovarian aging

In addition, the inducible demethylase JMJD3, which acts on H3K27me2 and H3K27me3, is a repressive epigenetic histone mark. Notably, transcription factors that transduce signals induced by inflammatory mediators and a variety of stress conditions can activate JMJD3 expression. JMJD3 induction is a key host defense response to environmental threats and cellular stress as JMJD3 promotes a wide range of genes, comprising pro-inflammatory agents and factors that inhibit cell proliferation. Also, JMJD3 can regulate inflammatory responses, including M2 polarization through STAT6 and control CD4+ T cell commitment by the factors of T-bet. P53 and INK4 box genes also have a role in the aging process as they are significant targets for the cellular senescence regulation that is JMJD3-induced. As the function of JMJD3 is enhanced by the RAS and p53 signaling, it can prevent cancer development by senescence that is induced by the oncogene. Excessive JMJD3 activation, on the other hand, can be harmful both through uncontrolled transcription and alteration of the nuclear structures due to H3K27me3 being a crucial epigenetic mark associated with the maintenance of the genome's 3D organization. The potentially opposing impacts of JMJD3, such as responses that are pro-inflammatory and anti-inflammatory, depending on the cellular setting are significant JMJD3 can create developmental responses in addition to causing cellular senescence, indicating that it is an epigenetic regulator at the intersection of inflammation and cellular senescence.

A schematic figure of the regulatory network associated with JMJD3.

Screening experiments have shown that stabilizing HIF-1 promotes the expression of specific KDMs. HIF-1α not only promotes KDM3A, KDM4B, KDM4C, and KDM6B expression, which improve gene transcription by the demethylation of H3K9 and H3K27 sites, but also KDM2B and KDM5B expression, which inhibit the transcription by demethylation of H3K4me2,3 sites. KDMs that are hypoxia-inducible promote the gene transcription stimulated by HIF-1α, but they can also affect the genome-wide chromatin landscape, particularly KDMs that are responsible of the H3K9 and H3K27 demethylation. Senescent cells have a deleterious effect on neighboring cells due to secreting inflammatory mediators as well as activating signaling pathways that stabilize the signaling of HIF-1α. Considering cellular senescence is an irreversible modification, it creates an inflammatory environment that activates HIF-1α inducible KDMs, putting adjacent cells at risk. This cycle is becoming more prevalent in the age-related diseases.