Dao-Zheng Yang*, Xin-Yuan Luan, Shu-Bin Wang

1 School of Life Science, Shandong University, Jinan, China; 2 Department of Surgery, Tianjin Nankai Hospital,Tianjin, China.

Introduction

Adult stem cells play essential roles in tissue self-healing and regeneration. Accumulating evidence shows that ageing is associated with the decrease and dysfunction of adult stem cells, which is thus considered to be one of important hallmarks of mammalian ageing [1]. Hematopoietic stem cells(HSCs), as one of adult stem cells, have been well functionally characterized recently. The capacity of long-term self-renewal and differentiation makes HSCs able to generate all mature lymphoid and myeloid lineages [2], including erythrocytes, granulocytes,platelets, macrophages and B- and T-lymphocytes. In young adults, it was shown that around 1000 active HSCs are responsible for the generation of all blood cell types [3], indicating a powerful self-renewal and differentiation potential. However, functions of HSCs are impaired with age (Figure 1), especially its ability to maintain the balance between self-renewal and differentiation, which is of vital importance to blood system [4, 5]. Compaired with its younger counterparts,aged HSCs show several characteristic phenotypes,including increased number, homing defect, impaired engraftment potential and repopulating capacity, and increased output of myeloid-biased progeny [6-10].

Figure 1 The characteristics of aged hematopoietic stem cells (HSCs).

Epigenetic regulations, including DNA methylation,histone modifications and non-coding RNAs interference, encompasses all heritable changes in gene expression that are not due to changes in DNA sequences [11, 12]. Such mechanism controls gene expression mainly via regulating chromatin structure and status, playing an important role in determining cells fate and ontogeny. Various epigenetic alterations has been recognized as another hallmark of mammalian ageing [1]. In the field of HSCs ageing,evidence shows that HSCs are not protected from ageing. Instead, loss of epigenetic regulation at the chromatin level may drive both functional attenuation of cells, as well as other manifestations of ageing,including the increased propensity for neoplastic transformation [5].

Traditional Chinese medicine (TCM) is a system of theories and therapies that was first documented in ancient Chinese classics dating back 2100 years.Epigenetics, combining genetics and environment,contributes to not only the stability of organisms but also their adaptability to the environment, which is consistent with the theory of human-environmental inter relation of TCM. Recently, epigenetics has been introduced to the area of TCM resulting in the hypothesis of an epigenetic role in the pharmacology of TCM prescriptions. Various traditional Chinese medicine has been shown to be safer and more effective for preventing cancer by targeting epigenetic landscape [13].

In this review, we aim to conclude that the altered functional potential of HSCs controlled at the epigenome level with age, focusing largely on DNA methylation, histone modifications, the two most characterized epigenetic marks. In addition, we will also talk about the epigenetic role of TCM in the treatment of hematological malignancies.

Epigenetic regulations and HSCs ageing

DNA methylation and HSCs ageing

DNA methylation is a potent epigenetic mark that promotes gene silencing. Methylation occurs at the 5-carbon position of cytosines (resulting in 5-methylcytosine or 5-mC) found in cytosine-phosphate-guanine dinucleotides (CpGs)through the action of the DNA methyltransferase(DNMT) family of proteins, including the de novo methyltransferases Dnmt3A and Dnmt3B and the maintenance methyltransferase Dnmt1.Dnmt1-deficient HSCs show self-renewal defects and decreased ability of multilineage hematopoiesis,indicating functions in both HSC self-renewal and differentiation [14, 15], while loss of Dnmt3A in HSCs impairs their differentiation and increases the number of bone marrow HSCs over serial transplantation [16].Interestingly, loss of Dnmt3B has minimal effects on adult HSCs function. However, a more severe block in differentiation occurred in HSCs when Dnmt3B was ablated combined with Dnmt3A. Furthermore, it was also reported that Dnmt3B has distinct functions, given that Dnmt3B accounts for some HSCs differentiation in the absence of Dnmt3A [17, 18].

Apart from DNMT family, recently TET2 (the Ten-Eleven-Translocation 2), a member of TET family that encode enzymes modifying DNA by hydroxylating 5-methylcytosine (5mC) [19], has been shown to be essential to HSCs self-renewal and differentiation. Consistent with this, somatic loss-of-function mutations in TET2 are frequently found in patients with myeloid malignancies, for example myelodysplastic syndromes. TET2 loss leads to increased size of HSCs pool and TET2-deficient HSCs were shown to develop enhanced stem cell self-renewal in vivo competitive transplant assays [20,21]. It was found that hypermethylation of Polycomb Repressive Complex 2 (PRC2) targets appears to accompany forced proliferation and ageing, suggesting that DNA methylation plays a critical role in regulating the physiological ageing of HSCs [22, 23].

Histone modifications and HSCs ageing

Histone modification is one of the major covalent modifications that occurs at histone tails, including methylation, acetylation, sumoylation, phosphorylation,and ubiquitination, which have a critical role in dynamic modulation of chromatin structure and function, contributing to the regulation of cellular gene expression. These modifications involve in the chromatin remodeling and impact DNA accessibility.By taking advantage of highly purified HSCs,genome-wide comparisons of histone modifications between young and aged mouse HSCs were performed,enabling us to have a comprehensive understanding of the link between histone modification and HSC ageing.Recently, three key regulatory chromatin marks,H3K4me3 (trimethylation of Lys 27 of histone H3),H3K27me3 and H3K36me3, were chosen to assess epigenetic alterations in young and old HSCs [24].Results from ChIP-seq suggest that aged HSCs exhibited broader H3K4me3 (an active mark) peaks,particularly in HSCs identity and self-renewal genes,showing a positive correlation with gene expression alteration. Furthermore, there was a strong positive correlation between changes of H3K4me3 and gene expression with age. Additionally, changes in H3K27me3 (a repressive mark) levels have also been described and similar to H3K4me3, H3K27me3 density around promoters expanded as well.Interestingly, p16INK4a（a tumor suppressor protein encoded by Cdkn2a）is repressed by H3K27me3 both in young and old HSCs, while the increase of Cdkn2a is thought to be a hall mark of ageing for virtually all tissues [1].

Polycomb group (PcG) proteins are key epigenetic regulators of HSCs fate by maintain and propagate regulatory histone modifications [25]. Two Polycomb repressive complexes (PRCs), PRC1 and PRC2, have been shown to have distinct functions in the control of HSCs self-renewal, with PRC1 crucial to maintain gene repression while PRC2 crucial to initiate gene repression. PRC1 is the main H2A ubiquitin (H2Aub)ligase [26]. Within PRC1, Cbx family members functions in the modulation of the balance between HSCs self-renewal and differentiation [27]. Via H3K27me3 binding, overexpression of Cbx7 enhances HSCs self-renewal，while overexpression of Cbx2,Cbx4 or Cbx8 contributes to differentiation. In addition, overexpression of another member of PRC1,BMI1, in cord blood leads to long-term maintenance of human hematopoietic stem/progenitor cells [28].

The PRC2 complex is responsible for H3K27me3 through its enzymatic subunits EZH1 and EZH2 [29,30]. PRC2 complex contains either EZH1 or EZH2,which is chromatin-modifying histone lysine methyltransferases [31]. Enforced expression of EZH2 in HSCs prevents their exhaustion during serial transplantations [32] and its conditional loss results in defect of muscle regenerative potential [33]. Much later it was shown that EZH2 in fact is frequently mutated in patients with myeloproliferative diseases.EZH2 deposits the epigenetic trimethyl mark that is recognized by the Cbx proteins contained in the PRC1 complex [27]. Whereas EZH1 and EZH2 have different chromatin binding properties, EZH1 can also provide enzymatic activity for the PRC2 complex. It was demonstrated that EZH1 is an important histone methyltransferase for HSCs maintenance [34]. EZH1 maintains repopulating HSCs in a slow-cycling,undifferentiated state, protecting them from senescence.Furthermore, Epigenetic and gene expression changes resulted from loss of EZH1 in aged HSCs showed that EZH1-mediated PRC2 activity catalyzes monomethylation and dimethylation of H3K27.

Members of the sirtuin family, NAD+-dependent protein deacetylases, particularly Sir2, have been investigated as potential anti-ageing factors. With seven homologs in mammals, the Sir2 family of histone deacetylases (HDACs) targets H4K16 and other proteins involved in regulating glucose and fatty acid metabolisms [35, 36]. Pharmacological inhibition of Cdc42 activity functionally rejuvenates aged HSCs,increases the percentage of polarized cells in an aged HSCs population, and restores the level and spatial distribution of histone H4 lysine 16 acetylation to a status similar to that seen in young HSCs [37], which leads to the hypothesis that epigenetic regulation by Sir2 family HDACs governed by the Cdc42 activity plays a role in HSC ageing [38]. Sirt3, a mammalian sirtuin, is highly enriched in HSCs [39]. Additionally,Sirt3 is dispensable for HSC maintenance at a young age but is essential at an old age. Importantly, Sirt3 upregulation in aged HSCs improves their regenerative capacity.

HSCs ageing and traditional Chinese medicine

HSCs typically show increased incidence of myeloid malignancies with age [40]. As pointed above,epigenetic alterations are considered as a hallmark of ageing and mutations in epigenetic regulator genes occur frequently in most hematological malignancies[41], with 20–22 % of de novo AML (acute myeloid leukemia) patients were found to have mutations in DNMT3A [42]. Simultaneously, epigenetic modifications are potentially reversible in contrast to genetic defects. In this context, remodeling of HSCs epigenome sheds a light on diseases preventive and therapeutic strategies. Indeed, 5-Azacytidine, a DMNT inhibitor, which epigenetically modulates various tumor suppressor genes, has been used for the myelodysplastic syndromes (MDS) and AML [43].Chemopreventive nutritional polyphenols, such as soy,genistein, resveratrol, catechin, curcumin, are currently evaluated for their ability to reverse adverse epigenetic marks in cancer (stem) cells to attenuate tumorigenesis-progression, prevent metastasis or sensitize for drug sensitivity [44].

Via a bioinformatic study, it was reported that 29.8%of 3294 TCM medicinals are epigenome- and miRNA-modulating by interacting with Polycomb group and methyl CpG-binding proteins [45].Strikingly, within 200 TCM formulas, 99% of them are epigenome- and miRNA-interacting. Some herbal medicines are reported to target epigenetic modifiers and hence contribute to inhibit the proliferation of cancer cells (Table 1). Feijoa sellowiana extract,particularly flavone, exerts anti-cancer activities on hematological cancer cells [46]. Accompanied by p16 overexpression in human myeloid leukemia cells,Feijoa apoptotic activity correlates with the induction of HDAC inhibition.

Trichosanthin [47], tanshinone IIA [48], arsenic trioxide (ATO) [49, 50], yugan granule [51] and genistein [52] have been reported to have anti-cancer effects by targeting DNMTs in various cancer cells.Within these medicines, western blot and immunohistochemical analysis confirmed that tumor suppressors including p16 were markedly enhanced after treatment with a low concentration of ATO in human liver cancer cells. Additionally, ATO decreased the mRNA expression of DNMT 1 and also dose-dependently inhibited DNMT activity.Collectively, a low concentration of ATO induces demethylation of tumor suppressor genes by inhibition of DNMT and reactivates the partially/fully silenced genes in liver cancer cells [49]. ATO is also considered to be an efficient drug for the treatment of acute promyelocytic leukemia (APL). Researcher showed that the extent of total genomic DNA methylation of HL-60 cell decreased after ATO treatment, which is accompanied by reduced expression of DNMT3B with DNMT1 no significant change [50].

Curcumin, which is found in turmeric, functions as a strong anticancer agent in human prostate cancer cells through the modulation of HDACs [53-55]. Although the total HDAC activity was decreased upon CUR treatment, such treatment showed different effects on the protein expression of HDACs, increasing the expression of HDAC1, 4, 5, and 8 but decreasing HDAC3. Further analysis showed that CUR decreased the enrichment of H3K27me3 at the Neurog1 (a cancer methylation marker) promoter region as well as at the global level. Triptolide, which is the principal active ingredient of Chinese herb Tripterygium wilfordii Hook.F, has various functions such as antitumor properties. In the field of hematology, triptolide was shown to be able to inhibit the proliferation of multiple myeloma cells in a time- and dose-dependent manner,with induced G0/G1 cell cycle arrest and apoptosis[56]. In addition, the possible anti-myeloma mechanism of triptolide was to decrease histone H3K9 and H3K27 methylation via the downregulation of histone methyltransferase SUV39H1 and EZH2,respectively. Interestingly, by modulating histone H3-Lysine 9 (H3-K9) methylation and deacetylation,genistein is able to activate expression of several aberrantly silenced tumor suppressor genes as well,indicating a broad effect of Chinese herb on epigenetic landscape [58].

Taken together, these results suggest another mechanism to develop effective therapeutics based on epigenetics, and offer a strong support for the proposition that we can treat hematological malignancies resulting from HSCs ageing by taking advantage of the epigenetic role of TCM pharmacology.

Table 1 The epigenetic regulations of TCM on tumors

Conclusion and perspectives

The role of epigenetic regulation in HSCs ageing is gradually becoming clearer. However, much work remains to be done to decipher the complete picture of epigenetic machineries that regulate HSCs ageing. In addition, HSC ageing is related to hematopoietic system malignancies. Therefore, targeting epigenetic genes may be a promising strategy to treat hematopoietic system malignancies. TCM is attractive to explore drugs targeting epigenetic modifiers.Whereas an increasing amount of TCM have been indentified to be effective in the treatment of various cancers in a epigenetic manner, more studies need to be carried out to assess the role of TCM in treating hematopoietic malignancies and other HSC-ageing-related diseases.

Competing interests

The authors declare that they have no competing interests.

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