Epigenetic Modifications Involved in Ageing Process: The Role of Histone Methylation of SET-Domain

Abstract

Ageing is characterized by the maintaining deterioration of homeostatic processes over time, leading to functional decline and increased risk of disease and death. Several distinct mechanisms underlying ageing have been reported and mounting shreds of evidence have shown that histone methylation, an epigenetic marker, regulates gene expression during ageing. Recently, SET-domain genes have gained attentions and have been identified as histone methyltransferase involved in ageing process. Deletion of these genes extends lifespan and increased oxidative stress resistance in Caenorhabditis elegans depends on the daf-16 activity in the insulin/IGF pathway. In this chapter, we propose to investigate the role of histone methylation in the process of ageing and oxidative stress with an emphasis on the role of set-18 gene in ageing process.

1. Introduction

Ageing is considered as a complex and multi-factorial biological process driven by diverse molecular pathways and biochemical events shared by all living organisms [1]. It is characterized by the deterioration in the maintenance of homeostatic processes over time, which leads to functional decline and the increased risks of diseases and death [2]. It is also known as a general and complex biological process that predisposes humans to many complex diseases, including neurodegenerative diseases, type 2 diabetes, and various types of cancers.

Numerous studies have focused on the decipherment of the hallmarks of ageing in order to identify potential therapeutic targets to mitigate the ageing process. These hallmarks include stem cell exhaustion, altered intercellular communication, senescence, genomic instability, and recently epigenetic deregulation [3]. The end result of epigenetic changes alters the local accessibility to the genetic material, leading to aberrant gene expression, reactivation of transposable elements, and genomic instability. DNA accessibility is a determinant of genetic expression in the human genome. Strikingly, certain types of epigenetic information can function in a transgenerational manner to influence the lifespan.

In eukaryotes, the physiological and cellular mechanisms of ageing are conserved. However, various molecular mechanisms of ageing come from a variety of eukaryotic models, like Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, mammalian tissue culture and human premature ageing [4]. Several distinct mechanisms underlying this process have been reported. Among them, the genetic screening and naturally occurring mutations have identified hundreds of genes involved in different pathways that affect ageing, of which insulin-like growth factor 1 (IGF-1) signalling, target of Rampamicyn (TOR) signalling, autophagy pathway, mitochondrial respiration signalling pathway, and hypoxia-inducible factor 1 (HIF-1) pathways [5].

Recently, researchers have tended to hold a comprehensive view to explain the complex interaction of genetic and environmental factors. Epigenetics, which can be defined as the study of stable genetic modifications that result in changes in gene expression and function without a corresponding alteration in the DNA sequence [6], has been found to be a necessary component to establish the overall understanding of ageing. Evidences have shown that epigenetic alterations can be regarded as a trigger of ageing pathway mechanisms, namely [7] histone modifications, which are supposed to be the essential component of epigenetic regulation and broadly studied. Nowadays, histone methylation occupies a crucial role during the development of organisms. Regulators of histone methylation have been mainly associated with ageing in worms and flies [8]. By RNAi screening of C. elegans genes, histone methyltransferase and demethylase genes have been identified as the key modulator of lifespan. These modifications are involved both in gene silencing, generally associated with transcriptional repression (H3K27me3), and in gene activation, associated with gene expression (H3K4me3) [9, 10], among them are SET-domain genes.

Since their discovery, SET-domain genes have attracted significant interest in multiple areas of biology and medicine, including endocrinology, growth, metabolism, nutrition, ageing, and oncology. The signalling pathways elicited by SET-domain genes have been extensively characterized in biochemical and molecular terms over the past years. However, fundamental questions regarding basic differences between the mechanisms of action of SET-domain genes and the closely related role in ageing are yet to be resolved. More recently, a study carried out by Su et al. 2018 [11] observed that a SET-domain protein, set-18, which is presented as a novel H3K36 dimethyltransferase in C. elegans, is specifically expressed in muscle, and its expression level is gradually increased during ageing. The mutation on this gene extended C. elegans lifespan and increased its oxidative stress resistance. However, the mechanism in which this gene is implicated remains unknown [4].

This chapter will provide a collection of information dealing with the role of epigenetic modifications involved in ageing process and highlights the significant role of histone methylation of SET-domain with an emphasis on the set-18 gene. Among all the epigenetics modifications affecting organism lifespan, the histone methylation and demethylation stand out as a highly conserved and critical mechanism.

2. The effect of histone modification in ageing regulation

Although many studies have shown the molecular mechanism of ageing and the regulatory pathways, it appears that just in the past decade scientists have begun to understand that dynamic histone modification may be involved in the regulation of the ageing process of organisms. Histone proteins, in contrast to DNA, are subject to a huge number of modifications that contain methylation, acetylation, ubiquitination, and phosphorylation [8]. Researchers have observed that histone modifications undergo alterations during the ageing process. Although these alterations are the causes or consequences of ageing are still debatable, it is widely accepted that there is a certain connection between them as shown in Figure 1.

Figure 1.

The interplay between histone modifications, oxidative stress, and ageing.

Recent findings revealed that epigenetic factors that regulate histone methylation, a type of chromatin modification, can affect the lifespan of organisms. While acetylation of histone tails is largely ephemeral in nature. Histone methylation is widely observed to be a mark that confers long-standing epigenetic memory [12, 13]. This histone modification is accomplished by the catalysis of histone methyltransferase (HMT). According to the different methylation sites, it is mainly divided into histone lysine modification and arginine modification. Histone lysine methylation occurs at three different levels: monomethylation modification, dimethylation modification, and trimethylation modification. This modification is highly conserved between single-celled organisms and different species of mammals [4].

Most of the known targeted lysine of histone methyltransferase occurs on histone H3, which thereby serves as a conduit of epigenetic regulation. Mostly, lysine methylation at histone H3, lysine 9(H3K9), H3K27 or H4K20 act as gene silencing, whereas H3K4, H3K36 or H3K79 are associated with the actively transcribed genes [14]. Histone methylations, especially histone 3 lysine 4 trimethylation (H3K4me3) activation and H3K27me repressing, are epigenetic modifications with close ties to transcription and have been directly linked to lifespan regulation in many organisms [15]. These alterations are not only hallmarks to monitor and evaluate the course of ageing, but also the potential targets of anti-ageing treatments.

The age-dependent variations of histone marks increase the instability of the genome and influence the expression of corresponding genes, and the accumulated genomic instability in old cells can lead to oxidative stress and lifespan reduction.

Mounting evidence has reported the correlation between oxidative stress, ageing and epigenetic modifications. To date, histone methylation is a classic epigenetic mark recognized to be involved in gene expression and has key functions in ageing control [16, 17, 18, 19]. By RNAi screening of C. elegans, histone methyltransferases and demethylases have been identified as key modulators of lifespan. These include H3K27 demethylase UTX-1. H3K9 trimethyltransferase set-26 and H3K4 trimethylation complex set-2/ASH2/WDR-5 [11] are SET-domain proteins.

3. Oxidative stress and ageing

Free radicals are highly considered as reactive atoms or molecules having one or more unpaired electron(s) in their external shell. These radicals are produced by losing or accepting a single electron, therefore acting as oxidants or reductants [20]. The terms reactive oxygen species (ROS) refer to reactive radical and non-radical derivatives of oxygen. They are produced by all aerobic cells in the mitochondria and play an important role in cell immunity, in ageing as well as in age-related diseases [21] and they are also known to cause oxidative damage to cells and molecules. This, in turn, is widely recognized as a determinant of both lifespan and health span.

Ageing, over time, is considered as a progressive loss of tissue and organ function. It is generally regarded as an endogenous, irreversible and deleterious process poorly understood biologically [22]. However, in spite of considerable research efforts, the endogenous causes of ageing remain elusive. More recently, the free radical theory of ageing, later termed as oxidative stress theory of ageing, has been postulated. This theory is based on the structural damage-based hypothesis that age-associated functional losses are the result of the accumulation of oxidative injury to macromolecules such as lipids, DNA, and proteins by ROS [22]. The theory was later refined by Harman himself to emphasise the role of mitochondrial ROS, as the majority of free radical oxygen species (ROS) production originates in the mitochondria of mammalian cells, and was termed as the mitochondrial theory of ageing (Figure 2). Even though the exact mechanism of oxidative stress-induced ageing has remained unclear, however, scientists are agreed that the increased ROS levels lead to cellular senescence, a physiological mechanism that stops cellular proliferation in response to damages that occur during replication [23].

Figure 2.

Oxidative stress implication in ageing.

4. SET-domain protein implication in ageing

SET-domain is a short name for a highly conserved 130 to 140 amino acid motif characterising a group of proteins known to methylate histones on lysine. The function of SET-domain proteins is to transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of a lysine residue on the histone or other protein [24].

Initially, the SET-domain proteins were associated exclusively with the regulation of developmental genes in metazoan. However, the finding of SET-domain genes in the unicellular yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that SET-domain proteins regulate a much broader variety of biological programmes [25]. By performing a targeted RNAi screen in fertile worms with selected genes that encode known worm methyltransferases, proteins containing the enzymatic domain of methyltransferases (SET-domain), or orthologues of regulators of histone methylation, the results depicted that set-9 and set-15 knock-down extended lifespan and also set-2 and set-4 knock-down extended fertile worm lifespan as previously reported by many investigators [8].

The authors also investigated whether the known histone lysine methyltransferase regulates lifespan and screened genes encoding the SET domain in C. elegans by RT-qPCR, and the results revealed that the mRNA levels of set-2 and set-15 increased significantly on day 11; both genes had been reported to promote ageing. In addition to these two genes, they found that the mRNA levels of set-10, set-18, set-32 and F54F7.7 were also upregulated in nematodes, and these genes have not yet been reported to be involved in lifespan regulation [11].

Focusing on set-18, results revealed that this gene expression increases according to animal age and is highly expressed in muscle. The mutation on this gene revealed a lifespan extension and oxidative stress resistance compared to the wild-type worms. It is important to note that, when there is increased exposure to reactive oxygen species (ROS), the cell enters the state of chronic oxidative stress. The more a cell is growing, the more oxidative stress damages are increasing; thus, this has an influence on longevity [3].

Studies have shown that there is a close relationship between the survivals of the mitochondrial and that of the cells due to the central role of mitochondria in programmed cell death (apoptosis) as well as the important involvement of ROS produced at 90% in mitochondria. High levels of ROS and calcium, acting together, can trigger the mechanism of cell death via apoptosis or necrosis. For several years, plenty of researches aimed to understand the adverse effects of ageing and were conducted out on a wide range of model organisms, and nine general hallmarks of ageing in living organisms have been identified. These hallmarks affect the organism at different scales. Some occur at the molecular level within cells, while others impact tissues (muscles) and even beyond at the level of an organ or the entire organism [3]. These mechanisms have been revealed to influence longevity partially dependent on daf-16, a prominent longevity gene that encodes the worm orthologue of the highly conserved forkhead transcription factor forkhead box O (FOXO).

Sequence analysis showed that set-18 has high homology to the mammalian histone methyltransferase SMYD family, which contains a SET-domain split into two segments by a myeloid, nervy, and DEAF-1 (MYND) domain [11]. set-18 contains a conserved SET-domain that encodes proteins homologous to human SMYD1, SMYD2, and SMYD3 [26, 27]. The human homologue SMYD1, SMYD2, and SMYD3 of set-18 have been reported to have the activity of histone methyltransferase. The SMYD family encloses five discrete proteins, of which SMYD1-5, with reported functions in both normal and pathologic conditions (ageing diseases). The key feature of all SMYD family members has been established to be the methylation of H3K4. For many SMYD family members, the SET-domain contains two sections: 1) the S-sequence, which may work as a cofactor binder as well as for protein–protein interactions and 2) the core SET-domain, which functions as the primary catalytic location [28, 29]. In close relation to the SET domain are two other domains: 1) the post-SET and 2) the SET-I, which assist in cofactor binding, substrate binding, and protein stabilization [30, 31].

5. set-18 gene involvement in ageing and lifespan regulation

C. elegans set-18 gene was identified as a histone H3K36 dimethyltransferase. The deletion of set-18 increased lifespan and oxidative stress resistance depending on the daf-16 activity in the insulin/IGF pathway. Muscle-specific expression of set-18 increased in aged worms, resulting in elevation of global H3K36me2 and inhibition of daf-16a expression and, consequently, decreased longevity. These results suggest that H3K36me2 and H3K36me3 modification have distinct functions in regulating ageing [11].

The FOXO family of transcription factors are highly conserved key converging points of several longevity pathways. C. elegans has a single FOXO orthologue, daf-16, and mutants with daf-16 deficiency exhibit shortened lifespan compared with wild-type animals. Very recent studies showed that daf-16 functions downstream of a histone modifier to influence lifespan. daf-16 lifespan epistasis analyses suggest that the set-18 gene works through daf-16 to modulate lifespan. As daf-16 transcript levels are not affected by set-18 status in worms and that set-18 regulates global histone modifications, an interesting possibility is that set-18 acts as a cofactor of daf-16 in target gene regulation via changes in local chromatin accessibility.

However, it is also possible that like UTX-1, set-18 modulates the lifespan by regulating the expression of components of daf-16-dependent longevity pathways. Several well-known longevity pathways impinge on daf-16, including the daf-2/insulin pathway (insulin and IGF-1 signalling), the germline pathway, and the energetic metabolism pathway. Lifespan and oxidative stress tolerance are promoted by the FOXO daf-16 and suppressed by its upstream IIS pathway genes such as the exclusive insulin-like growth factor receptor daf-2 [32, 33].

The insulin and IGF-1 signalling (IIS) pathway are so highly conserved to modulate ageing and longevity. The IIS pathway is a signal transduction cascade that consists of insulin-like peptides (ILPs), an insulin/IGF-1 receptor (daf-2), a phosphoinositide 3-kinase (AGE-1/AAP-1/PI3K), serine/threonine kinases (PDK-1, AKT-1 and AKT-2) and the pivotal downstream forkhead box O transcription factor (daf-16) in C. elegans. This cascade in turn phosphorylates the FOXO/daf-16 and prevents it from entering the nucleus to trigger anti-ageing genes, such as the genes conferring resistance to heat, oxidative stress resistance and DNA damage [24, 25, 26, 27] as presented in Figure 3. daf-16/FOXO receives phosphorylation from the direct upstream AKT kinases mediated signal transduction response to insulin or IGF and is subsequently sequestered in the cytoplasm by 14-3-3 proteins [34, 35, 36], which antagonises FOXO and negatively regulates the longevity [27, 32].

Figure 3.

Possible action of set-18.

6. Conclusion

As ageing is inevitably a biological process associated with the deterioration of functional activities and resistance to hazards of individuals, researchers are always interested in investigating the relevant factors affecting the ageing process. The continued combination of functional studies and molecular analyses in different age groups, different organisms, and different tissue types will hopefully provide the details necessary to comprehend this evolutionarily conserved fundamental process and to facilitate the development of therapeutic interventions to counteract age-induced complications or to explore the direction of anti-ageing or rejuvenation. Our chapter revealed the role of epigenetic modification in ageing. Changes in epigenetic status have been shown to associate with ageing in many organisms. We focused on the role played by various histone modifications especially on histone methylation to lifespan modulation. Much more attention was on the SET-domain containing genes, set-18; further, we have shown that this gene regulates lifespan through daf-16, which is implicated in various mechanisms underlying ageing and oxidative stress. We demonstrated that set-18 as an H3K36 dimethyltransferase is expressed specifically in muscle. Hence, their exact role in ageing and its underlying mechanism has yet to be explored, thus providing more directions and strategies for the rejuvenation of ageing and the recovering of age-related diseases.

Acknowledgments

The authors thank Professor Li Xiaoxue, Professor Mbemba Fundu, and Professor Iteku Bekomo Jeff for their advices and contribution.