Cellular stress and epigenetic regulation in adult stem cells

HSC

The lifelong self-renewal capacity of HSCs is crucial for blood regeneration and giving rise to immune cells. This is supported by their quiescent state, where HSCs remain dormant and are metabolically conservative, thus reducing the induction of intracellular stress. When needed, HSCs are required to respond quickly and efficiently to a given situation where increased blood demands and/or cellular output for various immune responses, such as inflammation caused by bacterial infection. With the ability to rapidly jumpstart the appropriate intracellular processes for myeloid- or lymphoid-directed differentiation, comes a price that exposes HSCs to replication stress considering the immediate demand for DNA replication and high turnover rate. This is why it is proposed that the stem cells will differentiate to a progenitor cell without self-renewing potential and the bulk of the demand is met by increasing progenitor cells when protecting the stem cell population. However, when HSCs experience acute/chronic replicative stress, conserved response pathways such as the ATR-mediated DDR and ATM-mediated apoptotic priming pathways are activated to prevent and resolve genotoxic insults that arise (96, 153, 154, 155). Epigenetic alterations and responses, such as histone modifications, have been studied to better understand and elucidate the cellular defense mechanisms that HSCs mount to resolve replication stress and the potential for insults negatively impacting genome stability and cell survival.

In HSCs from young (6–12 wk) and old (22–30 mo) C57BL/6 mice, a study compared the prevalence and impact of replicative stress in an age-dependent manner and investigated a novel function of yH2AX as an epigenetic regulator (29). Old cycling HSCs under ionizing-radiation-induced replicative stress in vitro displayed an accumulation of yH2AX foci without DDR activation accompanied by impaired S phase progression of the cell cycle, increase in senescence-associated β-galactosidase (SA-β-Gal) and Cdkn2a (p16) expression, decrease in DNA helicase components MCM4 and MCM6 gene/protein expression, and presence of chromosomal breaks. Induced replicative stress in young HSCs displayed similar effects and functional decline as old HSCs in vitro or after BM transplantation, however, to a lesser extent. To further elucidate the molecular mechanism(s) driving the observed effects, this study also demonstrated that yH2AX signals accumulated on ribosomal DNA (rDNA) in the nucleolus and was associated with decreased 47S rRNA transcripts and ribosomal biogenesis in quiescent old HSCs. This was not a result of replicative stress-induced mutagenic impact on rDNA, rather, it is suggested that nucleolar yH2AX acts as an epigenetic histone modification that represses rDNA gene expression because of failed dephosphorylation by yH2AX phosphatase PP4c as it was mis-localized in quiescent old HSCs, primarily being found in the cytoplasm. PP4c was found in both the nucleus and cytoplasm of cycling and quiescent young HSCs under replicative stress. Persistent yH2AX that arise from replicative stress thus may be a long-term or permanent histone modification silencing rDNA and downstream translation of critical regulators of HSC function and maintenance.

Ott1 (also known as RNA Binding Motif 15 [RBM15]), holds broad regulatory roles in hematopoiesis and is required to preserve HSC quiescence during replicative stress, but not steady-state conditions. This was found in Ott1 KO mice where HSCs derived from young mice displayed premature aging phenotypes such as higher sensitivity to replicative stress, increased myeloid lineage output bias, and increased expansion (90). Ott1 KO HSCs were also found to have a significant reduction in quiescent cells. Part of the protective role of Ott1 in replicative stress is its regulation of the Thpo/c-Mpl axis via c-Mpl H4 deacetylation and H3K4me3 involving histone deacetylase and methyltransferase Hdac3 and Setd1b, respectively (156) (Fig 2A). c-Mpl is a transmembrane hematopoietic cytokine receptor promoting quiescence or proliferation of HSCs in a Thpo concentration-dependent manner, to which it is Ott1-dependent.

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Figure 2. A schematic illustration of epigenetic changes in HSCs because of various stressors.

(A) HSCs under IR-induced replicative stress displayed an accumulation of yH2AX foci without DDR activation accompanied by impaired S phase progression, senescence markers, and a decrease in DNA helicase components. Ott1 maintains HSC quiescence during replicative stress, where HSCs exhibit premature aging phenotypes. EZH2 is down-regulated in murine HSCs under severe replicative stress, leading to stem cell exhaustion. CBP is a transcriptional coactivator and histone acetyltransferase involved in several processes, such as differentiation, proliferation, and gene regulation. (B) PRC1 and PRC2 complexes are key epigenetic regulators in gene silencing via histone modifications. Autophagy regulates oxidative stress by clearing mitochondria, but its repression increases hypomethylation in genes that accelerate myeloid differentiation. (C) Inflammatory signals reduce expression of the histone demethylase UTX/KDM6A, impairing DNA repair and increasing oxidative stress. Global gene expression analyses have identified down-regulation of SWI/SNF-related genes, which are involved in chromatin remodeling and transcriptional silencing. Inflamed HSCs show increased expression of P-selectin (Selp), driven by heightened NF-κB pathway activity, which up-regulates inflammation-related genes and contributes to HSC functional decline. Chronic inflammation also induces a senescence-associated secretory phenotype in HSCs, thereby perpetuating inflammation and impairing HSC function.

Polycomb group (PcG) proteins are well known as epigenetic histone modifiers (157) and are found to be involved in preventing and during replicative stress response (158, 159, 160). Enhancer of zeste homolog 2 (EZH2) is a PcG protein and well-characterized histone methyltransferase known for its role as a transcriptional repressor (89, 161). It is thought that replicative stress contributes to chromatin instability via the loss of histone methylation motifs that facilitate DNA condensation, among other factors (162). EZH2 was found to be heavily down-regulated in murine HSCs experiencing severe replicative stress because of serial transplantations when displaying signs of stem cell exhaustion (163). When EZH2 was overexpressed in exhausted HSCs, long-term self-renewal and fitness was restored, as was repopulation capacity.

The CREB-binding protein (CBP), and its paralog p300, have been well defined as crucial transcriptional coactivators and histone acetyltransferases in a wide array of cellular processes in development and adult tissue homeostasis such as stem cell differentiation, proliferation, and apoptosis (164, 165, 166, 167, 168). CBP regulates gene expression through two different mechanisms: (1) by acting as a scaffold for the recruitment of transcriptional complexes to RNA polymerase machinery and (2) direct involvement in protein and histone lysine acetylation (97, 164, 169). In HSCs derived from young Cbp conditional KO mice, conditional ablation of CBP was found to result in overall dysregulation of HSC homeostasis and reconstitution capacity when met with replicative stress as observed in a BM transplantation assay (170). These HSCs were found to display aberrant regulation of the cell cycle, where stem and progenitor cells largely remained in the quiescent state, and increased apoptosis in the LSK population, contributing to cellular exhaustion. Furthermore, a genome-wide analysis showed that CBP was bound to loci regions involved in several critical HSC transcriptional networks/regulators, including the Heptad network, members of the Kruppel-like factor family (Klf2, Klf10, and Klf13), Runx1, Gfi1b, and Gata2 (171). This was strongly associated with the down-regulation of Heptad target genes involved in HSC differentiation, cell-cycle regulation, and self-renewal in Cbp KO mice and overlap in genes bound by CBP and Heptad factors. This demonstrates the direct regulation of CBP on critical HSC-specific transcriptional networks that are critical for ensuring HSC survival and maintenance under stressed conditions. Considering its epigenetic function, CBP may modulate these networks and corresponding transcription factors via histone and protein acetylation. However, additional studies investigating the epigenetic influence and long-term alterations CBP has on HSCs in these networks and factors would need to be performed.

The current studies show that replicative stress can activate several factors to elicit epigenetic alterations primarily as histone modifiers for preserving HSC quiescence and normal function (Table 1). The roles of several histone modifiers previously mentioned hold cytoprotective roles in HSCs against phenotypes like what is seen in an aging context, namely increased expansion and myeloid-biased differentiation. Replicative stress-induced down-regulation of histone methyltransferase EZH2 led to chromatin instability via resulted HSC exhaustion. These histone modifiers are also involved in evolutionarily conserved replicative stress response pathways, such as DDR pathways. To our knowledge, there are no published studies reporting the effects of replicative stress on other facets of the epigenetic landscape, namely DNA methylation and noncoding RNAs, but these would be an area of interest to further investigate.

MSC

MSCs have not been reported to have extensive replicative stress, although autologous MSC-based studies have elucidated a protective role that epigenetic alterations have in aging MSCs in response to stress and other negative aging-associated phenotypes.

One of the common phenotypes of replication stress in adult somatic cells is cellular exhaustion induced by age-associated ER stress caused by impaired proteostasis via the accumulation of un- and misfolded proteins. This has been primarily observed in hMSCs in natural and premature aging disease models (100, 172). As a consequence of age-associated cell exhaustion and ER stress, organelle homeostasis becomes disrupted considering the ER is connected to or in contact with membrane-bound organelles such as the nuclear envelope (NE) or Golgi apparatus and mitochondria. However, the exact causal molecular mechanisms have yet to be fully understood. In vitro functional assays involving hMSCs demonstrated that ER stress sensing/ER transmembrane protein activating transcription factor 6 (ATF6) maintains organelle homeostasis and protects hMSCs directly from ER and, arguably, replicative stress (173). This was shown in CRISPR/Cas9-mediated ATF6^-/-–induced hMSCs from hESCs, where hMSCs displayed accelerated cellular senescence, structural dysregulation in the ER, NE, mitochondria, and heterochromatin. These effects were also present in FOS-deficient hMSCs, suggesting a critical role for FOS as a downstream mediator of ATF6 in ER stress response. FOS is a novel ATF6 target gene in hMSCs that encodes for a leucine zipper protein forming a transcriptional complex (AP-1) with factors of the JUN family. The epigenetic influence of ATF6 is present as seen in heterochromatin disorganization; however, the mechanistic role in this phenomenon was not elucidated. Considering the transcriptional regulatory role of ATF6 in ER stress response, additional genome-wide assays, such as chromatin immunoprecipitation (ChIP) and RT–PCR analysis, would help shed light on additional novel gene targets encoding direct epigenetic regulators.

Whereas MSCs are less prone to replicative stress, recent studies have found that age-associated replication stress-induced ER stress can cause accelerated cellular senescence, structural dysregulation of several organelles and, on the epigenetic level, heterochromatin in these cells (173). However, there are only a few studies that have reported replicative stress-associated epigenetic alterations, namely regarding histone accessibility and perhaps modifications (Table 1). Because of the lack of studies in this area, further studies are required to elucidate the mechanisms involved in the replicative stress-induced epigenetic alterations in MSCs and on other regulatory factors regarding all facets of epigenetics. This investigation would be of great interest for the current MSC therapies to address and resolve major challenges from cell isolation protocols to clinical application (174).

ISC

ISCs are required to self-renew and differentiate at a high rate to regenerate the intestinal epithelium to maintain proper function and, thus, are subjected to replicative stress. This replicative stress has been linked to genome-wide alterations to the DNA methylome in highly proliferative tissues, including the intestinal epithelium, where methylation levels increased (114, 115, 175, 176). The ISC compartment is also subjected to hypermethylation as demonstrated in a computational model of ISC DNA repair showing the indirect influence of stress response on DNA methylation (177). In silico simulations displayed that promoter regions marked with H3K27me3 were found to undergo DNA hypermethylation during DNA repair in response to replicative stress in moderately irradiated ISCs (Fig 4A). This was associated with an increase in de novo DNA methyltransferase (DNMT) activity recruited to open chromatin regions undergoing repair and containing low levels of DNA methylation. The hypermethylation of gene promoters in open chromatin regions remained stable even after DNA repair, thus inducing long-term alterations to the epigenome. Consequences associated with this alteration include the development of aging phenotypes and tumorigenesis (178, 179).

Investigating the influence of replicative stress on the epigenetic landscape within ISCs is of importance because of its subjectivity to this intercellular stressor. The above studies have reported that replicative stress-induced epigenetic alterations and effects on regulators have been observed on the DNA and histone levels (Table 1), but further investigation on their mechanisms and downstream effects is needed. At the DNA level, hypermethylation at promoter regions took place and remained stable even after DNA repair. This long-term alteration could allow for ailments such as cancer and age-associated phenotypes to develop over time. It is of interest to investigate stress response pathways such as those involved in DDR and tolerance that may induce an intracellular state (e.g., open chromatin) prone to de novo epigenetic modifications.

HSC

Oxidative stress has been causally linked to aging-associated phenotypes in HSCs, including an accumulation of DNA damage, apoptosis, telomere shortening, poor reconstitution capacity and loss of quiescence (91, 184, 189, 190). Within the last decade, there have been studies demonstrating the protective and maintenance role of conserved epigenetic regulators in HSCs, one of which involves PcG proteins.

PcG proteins are well known to be epigenetic regulators in gene silencing via histone modifications catalyzed by two major complexes, PRC1 and PRC2. PRC1 monoubiquitylates histone H2A at lysine 119 (H2AK119ub1), whereas PRC2 mono-, di-, and tri-methylates histone H3 at lysine 27 (H3K27me1/2/3) (191). Among the two PcG complexes, Bmi1, a subunit of PRC1, has been reported to have critical roles in maintaining the self-renewal and multipotent differentiating capacities in HSCs (192, 193, 194, 195) (Fig 2B). Collectively, it has been well established that Bmi1 is essential for the maintenance of normal HSC function under homeostatic conditions. In competition repopulation assays and in vitro studies, Bmi1 has been shown to confer protection against both replicative and oxidative stress in highly purified CD34⁻ LSK HSCs from Tie2-Cre;R26StopFLBmi1 mice where Bmi1 (92). In addition, purified CD34-LSK HSCs from Tie2-Cre;R26StopFLBmi1 mice cultured in buthionine sulfoximine, for oxidative stress induction, showed significantly more colonies than control HSCs with buthionine sulfoximine. This suggests that Bmi1 can enhance and maintain the self-renewal capacity as part of its cytoprotective role against oxidative stress. Intracellular ROS levels were also measured and found to be similar in HSCs and downstream progenitors between those with overexpressed Bmi1 and the control. From this observation, Bmi1 could then be described to confer resistance to oxidative stress in HSCs not by regulating the production/accumulation of ROS, rather through some unknown mechanism that allows HSCs to tolerate high levels of ROS. This is an interesting contrast to another study reporting that Bmi1 is essential for regulating a set of genes critical for mitochondrial function and ROS production (196).

The mitochondria are a known origin of ROS as they facilitate oxidative metabolism. If left metabolically active without regulation, aging-associated phenotypes such as altered fate decisions and dysregulated self-renewal maintenance may occur in HSCs. This is especially detrimental to the HSC compartment as it is necessary to largely remain quiescent to retain lifelong functionality. Autophagy has been reported to have an active role in regulating oxidative stress through suppressing metabolism by clearing mitochondria in young and old HSCs from mice (197). When autophagy was repressed, qRT-PCR and gene ontology analysis in HSCs showed a significant increase in hypomethylation in regions expressing genes involved in fate decisions upon the activation of oxidative metabolism. This was associated with accelerated myeloid differentiation, a known aging hallmark in HSCs. Interestingly, when comparing populations of old HSCs, only a small subset can activate autophagy despite an overall competency for autophagy induction. In consideration of these findings, age may be associated with lessened resistance against oxidative stress in older HSCs, causing long-term alterations in the methylome that confers aging-associated phenotypes.

There are clear indications of altered epigenetic mechanisms both in response to and a result of oxidative stress, under certain conditions such as aging (Table 1). Bmi1, a subunit of the histone modifier PRC1, displayed cytoprotective roles in enhancing tolerance against oxidative stress in HSCs. Its mode of action pertains to the maintenance and enhancement of HSC self-renewal capacity; however, the mechanism behind this is unknown. Investigating this pathway and the role of PRC1 in modifying the ubiquitination landscape among histones would be is warranted. Conversely, when left to its own devices, oxidative stress is shown to induce hypomethylation in regions promoting myeloid differentiation, thus altering HSC fate decisions towards the myeloid lineage similar to changes in the aged HSCs compartment where there is an expansion of myeloid biased stem cells (101, 198). These studies have only unveiled replicative stress-induced epigenetic alterations on the DNA and histone levels and its regulators. Thus, it would be of interest to elucidate the molecular mechanisms and additional potential epigenetic regulators involved in these long-term insults to the epigenome during oxidative stress for the regenerative potential of BM transplantation. In addition, investigating the cytoprotective roles of epigenetic regulators in response to oxidative stress would identify targets for epigenetic therapies involving hematopoietic diseases, such as leukemia.

MSC

MSCs have widely demonstrated its therapeutic potential in tissue repair and overall regenerative capacities in transplantation studies. However, transplantation and prolonged culturing of MSCs is associated with the induction of oxidative stress, resulting in MSC exhaustion and accelerated aging (102, 103).

Emerging evidence has reported the role of epigenetic regulations and alterations contributing to oxidative stress or mounting antioxidant defense mechanisms against it. In human endometrium-derived MSCs (hMESCs), prolonged DDR activation and increased yH2AX foci because of ROS-induced DNA damage were observed (103) (Fig 3A). It is the first study to report a molecular mechanism driving premature senescence of hMESCs under oxidative stress. hMESCs exposed to sublethal oxidative stress exhibited premature senescence, which is suggested to be attributed to increased p38MAPK/MAPKAPK-2 pathway activation. The downstream effects of p38MAPK activation involved permanent ROS production, resulting in prolonged DDR signaling. DDR activation is known to induce phosphorylation of yH2AX in mammalian cells and tissues (199). It could then be said that oxidative stress is causally linked to yH2AX formation as a potential epigenetic histone modification, simply rather than a DNA damage marker. To address this notion, further investigation showing overlap of yH2AX as an epigenetic regulator of rDNA or novel transcriptional regulatory roles is needed.

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Figure 3. A schematic illustration of epigenetic changes in MSCs because of various stressors.

(A) In MSCs, ROS-induced DNA damage induced prolonged DDR activation and increased yH2AX foci driving premature senescence. In addition, miR-210, miR-29a-3p, and miR-30c-5p confer resistance to oxidative stress in MSCs, although upstream regulatory mechanisms of these miRNAs require further investigation. In aging MSCs, oxidative stress inhibits osteogenic differentiation by increasing EZH2 levels, which elevate H3K27me3 at the Foxo1 promoter. (B) Dnmt3b was found to bind to the Shh promoter region and directly catalyze hypermethylation. HDAC1 negatively correlates with osteogenesis by deacetylating the Jag1 promoter in BMSCs, down-regulating JAG1 and the NOTCH signaling pathway, whereas mechanical stimulation reduces HDAC1 levels, rescuing JAG1-mediated osteogenesis. (C) Histone variant mH2A1.1 modifications play a significant role in epigenetic regulation via the TLR4 pathway, inducing SASP. Increased H3K9me3 in aged MSCs leads to a repressive chromatin state, cellular senescence, reduced self-renewal capacity, and elevated expression of aging-related genes such as Cdkn2a (p16) and Cdkn1a (p21), with KDM4B loss exacerbating these effects by raising H3K9me3 and HP1α levels, ultimately impairing MSC functionality and regenerative capacity. Overexpression of AHCY leads to genome hypermethylation, whereas CBX3/HP1γ contributes to increased levels of H3K9me3.

microRNA-210 (miR-210) has been reported to confer resistance to oxidative stress in MSCs both in in vitro and in vivo settings. In functional assays on cultured MSCs isolated from the BM of Sprague-Dawley rats, overexpression of miR-210 resulted in a significant reduction in apoptosis and an increase in cell viability under oxidative stress in a concentration-dependent manner (200). This was accompanied by an increase in SOD activity and a decrease in malonaldehyde and ROS concentrations, all of which are reliable markers of oxidative stress (201, 202, 203). miR-210 was found to be a key activator in the c-Met pathway, of which it acts upstream of ROS generation. Under oxidative stress without miR-210 overexpression, c-Met was found to be repressed, resulting in increased levels of ROS. However, the exact mechanisms were not addressed. This suggests that ROS acts as a suppressor of the c-Met pathway, thus down-regulating downstream targets involved in clearing ROS. However, upon robust miR-210-mediated activation of c-Met, antioxidant defenses may be mounted. Both in vitro and in vivo studies using mice with acute liver failure and isolated human adipose-derived MSCs reported that under oxidative stress, miR-210 is naturally up-regulated from basal levels to maintain normal MSC function and clear cellular and mitochondrial ROS through ROS inhibitor iron-sulfur cluster assembly protein 1/2 (ISCU1/2) (104). Through treatment with the small molecule zeaxanthin dipalmitate (ZD), basal levels of miR-210 were increased and maintained at relatively high levels through inhibition of the PKC/MAPK/ERK pathway, which enhanced cell survival and resistance to oxidative stress in both in vitro and transplantation studies. Although pharmacological overexpression of miR-210 shows positive clinical application in enhancing MSC-based transplantation therapies, further investigation into the natural regulation of miR-210 under oxidative would be interesting.

Additional miRNAs, namely miR-29a-3p and miR-30c-5p, have also been shown to confer resistance and cytoprotection under oxidative stresses through computational and experimental approaches. Global depletion of miRNAs performed in hMSCs through silencing DiGeorge syndrome critical region 8 (DGCR8) gene, which is essential for miRNA biogenesis (105), resulted in impaired stem cell function, proliferation, premature senescence, and increased ROS production (204). Computational and in vitro analysis of antioxidant function and regulation revealed decreased SOD2 expression because of DNMT3A-mediated hypermethylation of upstream regulatory regions with respect to the SOD2 gene. Furthermore, miRNAs miR-29a-3p and miR-30c-5p demonstrated direct regulation of DNMT3A, to which overexpression of these miRNAs rescued hMSCs from oxidative stress and premature senescence. This suggests a novel stress response pathway against damaging ROS levels involving the miR-29a-3p/miR-30c-5p/DNMT3A/SOD2 axis. The regulation of these miRNAs, however, was not addressed. Further investigation on the upstream regulatory mechanisms controlling miRNA levels would further establish and elucidate this novel oxidative stress response axis.

Downstream effects of oxidative stress were found to extend to mitochondrial DNA (mtDNA) on an epigenetic level. In young human heart MSCs under replicative and oxidative stress-induced premature senescence, mtDNA methylation increased considerably, whereas subsequent COX2 gene expression decreased (205). Cytochrome c oxidase (COX) (also known as complex IV) catalyzes the reduction in oxygen to water in the electron transport chain. COX2 is one of three core mtDNA-encoded subunits forming complex IV (106). Given its enzymatic function, decreased expression of COX2 would induce mitochondrial dysregulation and allow for diatomic ROS accumulation. The mechanism in which COX2 mtDNA methylation occurs was partially elucidated through the treatment of 5-aza-2′-deoxycytidine (AdC), an inhibitor of DNMT1. Upon DNMT1 inhibition, COX2 mtDNA methylation decreased with subsequent increased protein expression. This was not surprising considering that DNMT1 contains a mitochondrial targeting sequence (206). The exact mechanistic function of DNMT1 in mtDNA methylation was not addressed nor was the association of the specific methylated CpG COX2 sites to COX2 suppression and mode of regulation. A functional analysis demonstrated a causal link between oxidative (and replicative) stress to epigenetic alterations taking place on the mtDNA methylome, specifically to the COX2 gene. Further investigations on the molecular mechanisms and associations of key epigenetic regulators, such as DNMT1, to mtDNA under stressed conditions are warranted.

On the flipside of downstream epigenetic alterations and responses to stress, upstream phenomena are also critical in elucidating the crosstalk between the epigenome and stress. In aging BM-derived MSCs, oxidative stress inhibits osteogenic differentiation through Wnt gene suppression in a mouse model of osteoporosis facilitated by increased levels of EZH2, a conserved methyltransferase of histone H3K27 trimethylation (207). Aging MSCs that also displayed EZH2 overexpression had higher levels of ROS and conversely displayed a significant reduction because of EZH2 knockdown. Through CHIP and RT–PCR analysis, EZH2 was shown to increase H3K27me3 levels in the promoter region of Foxo1 and was supported by lower expression levels. FOXO1 is a critical transcriptional regulator of antioxidant agents such as CAT and SOD2. Thus, EZH2 elicits oxidative stress by allowing ROS levels to increase as a result of down-regulating FOXO1-mediated antioxidant agents. EZH2 perhaps could become an effective pharmacological target for epigenetic therapy in reversing oxidative stress induced by varying factors outside of aging.

In clinical settings, MSCs are highly regarded for their therapeutic potential as previously mentioned. However, protocols in the expansion and usage of MSCs need further optimization because of their being prone to oxidative stress (102, 103). The current studies show potential means for improving MSC survivability and viability via ROS reduction, namely through miRNA induction as reported above (Table 1). It would be of interest to further investigate the mechanism in the biogenesis and downstream effects of these miRNAs in MSCs undergoing oxidative stress. In addition, functional and transplantation assays involving external induction of these miRNAs in primary MSCs would be critical for gauging its efficacy and practical usage. In addition, external inhibition of epigenetic regulators promoting ROS production such as EZH2 (207), could prove to be an effective means of reducing oxidative stress when expanding and transplanting MSCs in a clinical setting (Table 1). These studies warrant further investigation into these epigenetic factors and identifying others.

NSC

Adult neurogenesis, which is essential for brain function, is characterized by a high rate of energy consumption and thus leads to the accumulation of ROS. Effective clearance of ROS is widely recognized as one of the critical factors in promoting brain rejuvenation and preventing disease progression (208). Conversely, accumulation of ROS promotes DNA damage and cell death, which may contribute to the pathogenesis of neurodegenerative diseases such as AD (88, 209).

One of the causes of significant increases of ROS production in NSCs is mitochondrial dysfunction. It has been suggested that this dysfunction, which results in increased ROS and impaired mitochondrial respiration, underlies defects in adult neurogenesis during aging (116). Some have suggested that antioxidant compounds (e.g., docosahexaenoic acid) may abate the adverse effects of ROS and, therefore, improve adult neurogenesis and delay the onset of neurodegeneration (208, 209). Oxidative stress sensing also has a role in regulating the balance between self-renewal and differentiation of NSCs. Namely, whereas increased ROS levels are typically associated with NSC proliferation and differentiation, recent findings showed that quiescent NSCs in the hippocampus exhibited the highest levels of ROS, adding a level of complexity to the relationship between redox regulation and NSC fate decision (210). Oxidative stress also up-regulates SIRT1, which can suppress NSC self-renewal and shift their fate towards the astroglial lineage (210). To improve the cognitive healthspan, it would be interesting to examine how compounds that are explored in the geroscience context, such as spermidine and fisetin, and which reduce oxidative stress and neuroinflammation, act specifically on NSCs.

ISC

Studies investigating the epigenetic response or modulations in ISCs under oxidative stress are scarce. This itself warrants further investigation considering the causal and protective roles of several epigenetic regulators seen in other stem cell compartments. In support of this, Park et al were the first to show direct evidence of age- and oxidative stress-induced accumulation of DNA damage in ISCs, alongside the use of yH2AvD, analogous to mammalian yH2AX, as a biomarker (211) (Table 1) (Fig 4B). Several studies have shown yH2AX as a short- and long-term epigenetic histone modification recruiting chromatin remodeling proteins for increasing chromatin accessibility during DNA damage repair (212, 213, 214). As mentioned previously, in old cycling HSCs, yH2AX was reported as a long-term histone modification suppressing rDNA and subsequent ribosome biogenesis (29). Collectively, yH2AX, and perhaps its analog yH2AvD, displays an epigenetic role largely under stress-induced DNA damage in somatic cells, including stem cells. Further investigations to elucidate this notion would be needed.

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Figure 4. A schematic illustration of epigenetic changes in ISCs because of various stressors.

(A) Replicative stress has been linked to genome-wide hypermethylation to the DNA methylome in the intestinal epithelium. Simulations displayed that promoter regions marked with H3K27me3 were found to undergo DNA hypermethylation during DNA repair in response to replicative stress in irradiated ISCs. This was associated with an increase in DNMT activity recruited to open chromatin regions undergoing repair and containing low levels of DNA methylation. (B) ISCs under oxidative stress demonstrate age- and stress-induced DNA damage accumulation in ISCs using yH2AvD which suppresses ribosome biogenesis. (C) Signaling molecules such as Wnt, Bmp, and Notch, produced by Paneth cells, regulate ISC fate and function by allowing Atoh1 transcription factor to bind, enabling cells to revert to a primitive state. Epigenetic modifications such as the incorporation of histone variant H2A.Z mediated by Znhit1, are central to ISC plasticity, influencing key genes such as Lgr5 and TGF-β crucial for self-renewal and differentiation, with specific roles for MTG8, MTG16, and Id3 in modulating chromatin accessibility and maintaining ISC identity.

MuSC

Epigenetic regulation of MuSCs is required to maintain stem cell homeostasis and myogenic differentiation potential for skeletal muscle repair. Part of this process, and during endurance exercise, involves a slight increase in skeletal muscle ROS production for ensuring healthy mitochondrial function and adaptations (215). However, excess ROS production may still arise and induce oxidative stress (119). Bmi1 is shown to maintain MuSC pools and myogenesis during muscle via repression of the ink4a locus (216, 217) after muscle injury and during oxidative stress. In addition, Bmi1 overexpression in mouse models of dystrophinopathies and DMD patients induced enhanced metallothionein 1 (MT1)-mediated protection against oxidative stress, which improved muscle strength and regeneration (218, 219). Functional analysis on MT1-mediated oxidative stress displayed increased ROS and 8-OHdG in cultured MuSCs of MT1 knockdown mice when compared with controls. The mechanism underlying the observed MT1-mediated protection against oxidative stress has not been addressed and warrants further investigation. Bmi1 is involved in gene silencing through histone modifications as an integral component of PRC1, which suggests that MT1 enhancement is a result of repressing a MT1 repressor (Fig 6A). In MuSCs of DMD patients and quiescent cells, Bmi1 and MT1 expression levels were reduced both at the protein and RNA levels (219). Increased Bmi1 was associated with an enhanced energetic state, marked by increased ATP production during in vitro and in vivo studies of human DMD MuSCs (219). Interestingly, ROS generation did not increase, possibly because of MT1- and PRDX2-mediated antioxidant activities. PRDX2 is an antioxidant enzyme that scavenges cellular peroxides and facilitates redox homeostasis (220). Bmi1 confers resistance to oxidative stress in MuSCs, perhaps through gene silencing of repressors and/or transcription factors contributing to increased mitochondrial ROS generation.

As a result of cellular maintenance and myogenic differentiation, ROS levels in MuSCs are allowed to increase without passing a certain threshold that would otherwise induce oxidative stress. In response to increasing ROS levels, the Bmi1 has shown to facilitate histone modifications which in turn mounts antioxidative mechanisms conferring resistance to oxidative stress (Table 1). However, its direct role and mechanism has yet to be elucidated. This warrants further investigation into the role of histone modifiers, and other epigenetic regulators, in response to rising ROS levels and effects on antioxidative mechanisms.

MSC

Skeletal loading (increased mechanical stress on bone) has been shown to be essential in regulating BM MSCs (BMSCs) for osteogenic differentiation during bone repair, development, and mineralization (107, 222, 223, 224). Epigenetic modifications involving DNA methylation and histone modifications are reported to regulate osteogenesis in response to mechanical stimulation in different manners.

In vitro studies on mouse MSCs cultured in media conditioned from mouse osteocyte cultures shortly exposed (24 h) to fluid shear stress (a proxy for mechanical stress) displayed a decrease in DNA methylation in adipogenic and osteogenic markers by at least 25% and 30%, respectively (225). Although genes within these regions become more accessible, transcriptomic analysis revealed increased expression in only late osteogenic markers and little to no change in early osteogenic markers and overall adipogenic markers. DNA methylation decreased to a higher degree in late osteogenic markers, allowing for its expression. It is possible that downstream pathways of mechanical stress prime MSCs for osteogenic differentiation through expression of late markers and increased gene accessibility in early markers via decreased DNA methylation. The connection between mechanical stress and direct effects on epigenetic modifications is intriguing, and it will be interesting to better understand these relationships.

Additional osteogenic-promoting DNA regions in BMSCs also underwent hypomethylation upon mechanical stimulation as shown in in vivo and in vitro studies (226). In BMSCs of young mice, mechanical stimulation via cyclic mechanical stretch was shown to activate the Hedgehog (Hh) signaling pathway, demonstrating a role as an endogenous mediator for promoting osteogenesis. Under normal conditions, methylation of the promoter and enhancing regions of Sonic hedgehog (Shh), a direct upstream activator of the Hh signaling pathway, was present, resulting in gene silencing (227, 228). Through ChIP analysis, Dnmt3b was found to bind to the Shh promoter region and directly catalyze hypermethylation. However, mechanical stimuli on BMSCs demonstrated direct down-regulation of Dnmt3b protein levels accompanied by downstream up-regulation of Shh and subsequent activation of Shh-mediated osteogenesis. Regarding Shh demethylation, no mechanism nor epigenetic regulator was addressed. Ten-eleven translocation (TET) enzymes are conserved dioxygenases that mediate DNA demethylation and have been reported to regulate osteogenic differentiation in MSCs by increasing chromatin accessibility (229). TET-mediated demethylation possibly has a direct role in Shh up-regulation by reversing Dnmt3b-mediated methylation.

Histone deacetylases (HDACs) are negatively correlated with osteogenesis, namely with the expression of osteogenic markers in MSCs (112, 230). In BMSCs of mice and healthy human donors, HDAC1-mediated histone H3 deacetylation of the Jag1 promoter was present, resulting in down-regulation of JAG1 (231). JAG1 is a direct activator of the NOTCH signaling pathway and is essential for the induction of osteogenic differentiation in BMSCs (232, 233). The downstream effects of JAG1 down-regulation included the repression of the NOTCH signaling pathway and ultimately inhibited osteogenesis (Fig 3B). Mechanical stimulation via cyclic mechanical stretching enhanced osteogenesis, even in mice that underwent skeletal unloading. ChIP analysis revealed that mechanical stimuli directly down-regulated HDAC1 protein levels, which in turn rescued JAG1-mediated osteogenesis. This was supported by increased levels of osteogenic markers including ALP, COL1a1, and OCN.

BMSCs have demonstrated they maintain stem cell identity through epigenetic repression via DNA methylation and/or histone H3 deacetylation of promoter regions belonging to osteogenic differentiation-promoting genes (Table 1). Upon mechanical stimulation, the reversal of these epigenetic modifications is elicited through direct regulation of their respective modifiers, which is critical for bone development and formation. As such, hypomethylation and acetylation acts as an “ON” switch for osteogenesis in BMSCs upon mechanical stress whereas hypermethylation and deacetylation act as an “OFF” switch. In the current literature, mechanical stress-induced hypomethylation and acetylation were reported to have conferred osteogenic priming in MSCs (Table 1). It would be of interest to further investigate the mechanism involved in the interplay between these epigenetic alterations for developing interventions against bone-weakening ailments (e.g., osteoporosis). These epigenetic regulators also show potential as targets for enhancing BMSC-based transplantation or direct pharmacological manipulation.

MuSC

Skeletal muscle loading (increased mechanical stress on skeletal muscle tissue) activates the regulatory networks governing myogenic differentiation of MuSCs, which in turn can induce an anabolic hypertrophic response, enhanced tissue function, and overall muscle regeneration (234, 235). Epigenetic regulators involved in maintaining stem cell identity or promoting myogenic differentiation have been reported to be differentially expressed upon exposure to mechanical stress.

In vitro and in vivo analysis using adult mice revealed protein arginine methyltransferases 5 and 7 (PRMT5/7) to promote MuSC self-renewal and myogenic differentiation during muscle regeneration (120, 121). Arguably, mechanical stress may be strongly connected in consideration that strenuous mechanical activities such as weightlifting are well-established inducers of muscle regeneration (122). PRMT5/7 catalyzes the transfer of methyl groups onto arginine residues of proteins, including histones (123, 124, 236, 237). The inactivation or KO of PRMT5/7 in adult MuSCs underwent cell-cycle arrest and premature senescence, which in turn inhibited muscle regeneration. CHiP analysis revealed a direct role of PRMT5 in repressing p21 expression via histone dimethylation of H3R8 (H3R8me2) in two of four regulatory sites upstream of the murine p21 gene: upstream enhancer-like region (En) and p53 binding site (p53BS). However, inactivation of p21 in MuSCs of Prmt5 KO mice resulted only in increased MuSC proliferation and number of myogenic colonies, but it did not fully restore muscle regenerative capacity. This suggests that PRMT5 is involved in an epigenetic regulatory network governing several facets of MuSC function and maintenance outside of p21 regulation. Differing from PRMT5, PRMT7 regulates p21 expression via DNMT3b-mediated hypermethylation of two CpGs at the p21 promoter (Fig 6B). In addition, Dnmt3b and p21 promoter regions exhibited decreased H4R3me2 levels and a decrease and increase in the activation mark H3K4me3, respectively. However, a CHiP analysis of PRMT7 at each promoter site was not performed, which warrants further investigation into a possible direct regulatory role for p21 expression.

The epigenetic regulatory roles of PRMT5 and 7 in promoting muscle regeneration displayed a contrasting interplay between histone methylation sites of tumor suppressor genes p21 and p53 and Dnmt3b promoter regions (Table 1). In addition, PRMT7 facilitated DNMT3b-mediated DNA hypermethylation of the p21 promoter region. The epigenetic mechanisms displayed during muscle regeneration were not addressed under mechanically stressed conditions However, it is arguable that these epigenetic alterations behave similarly, if not the same, in MuSCs under direct mechanical stress as muscle regeneration takes place after mechanically stressed muscle after strenuous exercise and muscle tissue hypertrophy (122). It would be intriguing to perform additional studies confirming this notion. The effects of mechanical stress on MuSCs have been widely studied (238); however, studies regarding its influence on the epigenetic landscape are scarce and would be of interest to further investigate.

HSC

Inflammation serves as a key trigger for activating dormant HSCs during infections or other hematological stresses (241). Immature HSCs typically in a quiescent state, can be prompted to proliferate by inflammatory stimuli, acting as a reserve directed to produce mature blood cells during stress (242, 243). However, prolonged chronic or acute inflammatory stress can debilitate HSCs, leading to reduced regenerative ability and a bias towards myeloid cell differentiation, thereby impacting long-term function (244).

HSCs are critically influenced by inflammation and infections, impacting their functionality, differentiation, and long-term viability. Acute inflammation is typically a transient response aimed at restoring tissue homeostasis, but chronic inflammation can disrupt HSC homeostasis. Infections and persistent inflammatory signals can lead to an overproduction of myeloid cells at the expense of lymphoid cells, which skews the differentiation of HSCs. This myeloid bias, often observed in aged individuals, is driven by pro-inflammatory cytokines like IL-1, IL-6, TNF-α, IFN-α, and IFN-γ (93, 126, 245, 246, 247, 248). These cytokines can disrupt the BM niche by altering the interactions between HSCs and their supportive microenvironment. For instance, IFN-γ can displace HSCs from quiescence-promoting CAR cells via increased BST2 expression (95). This relocalization reduces the influence of quiescence maintaining signals from CAR cells, prompting HSC activation and increased differentiation. The BST2 interaction enhances HSC binding to E-selectin, facilitating movement within the BM and subsequently more prone to activation and depletion. In addition, IL-1 significantly accelerates HSC differentiation, as demonstrated by the faster division rates and enhances expansions of HSCs treated with IL-1 (248). This effect is mediated through early activation of the transcription factor PU.1, which drives myeloid lineage commitment. The study goes further to show that, chronic exposure to IL-1 results in a sustained increase in myeloid cell production at the expense of other lineages but compromises the long-term regenerative capacity of HSCs. Continuous activation through inflammatory signals can exhaust HSCs, reducing their ability to stay quiescent, self-renew, and maintain long-term hematopoiesis.

Specific epigenetic regulators are affected by inflammatory signals, exacerbating the aging and functional decline of HSCs. The histone demethylase UTX/KDM6A, which maintains HSC potential decreases in expression with age and inflammation. This reduction leads to an aged gene expression profile in HSCs, characterized by impaired DNA repair mechanisms and increased oxidative stress (94). Mice with deficient UTX exhibited trilineage dysplasia, a condition such as myelodysplastic syndrome (MDS) in humans. Inflammatory cytokines can also influence the activity of enzymes such as DNMT3A and TET2, which are crucial for DNA methylation and demethylation processes. For example, elevated levels of TNF-α enhances the proliferative advantage of TET2 deficient HSCs (249), and IFN-γ can drive expansion of DNMT3A knockout cells (250). Mutations in these genes are commonly found in clonal hematopoiesis and are associated with an increased risk of hematological malignancies. Inflammatory conditions can further promote the expansion of these mutated HSC clones, increasing the likelihood of disease progression.

One notable change is the increased expression of P-selection, a cell surface adhesion molecule associated with physiological stress, including inflammation and aging (251). In HSCs, there is a substantial rise in P-selectin (Selp or CD62P) levels on the surface of aged and inflamed HSCs (98, 252). With age, HSCs exhibit increased expression of P-selectin, which typically is exclusive to activated platelets and endothelial cells during an inflammatory response (99). This suggests that HSCs are exposed to a heightened inflammatory state within the BM. The NF-κB pathway, a critical mediator of inflammation and selp expression, is significantly activated in aged HSCs, as evidenced by enhanced nuclear localization of the p65 subunit older cells (253). This activation results in the up-regulation of several inflammation-related genes, which further perpetuates the inflammatory state and contributes to overall functional decline in HSCs (Fig 2C). Another striking example is the inappropriate expression of the IgK gene in aged HSCs, which is not normally active in these cells but becomes highly expressed because of a loss of epigenetic regulation at the IgK locus. Furthermore, global analyses of gene expression have identified significant down-regulation of genes involved in chromatin remodeling and transcriptional silencing, such as the SWI/SNF-related chromatin remodeling genes (253, 254). This lack of regulation suggests a broader loss of transcriptional control across the genome, leading to inappropriate gene activation and increased risk of genomic instability.

Lastly, chronic inflammation affects HSCs through the induction of a senescence-associated secretory phenotype. Senescent HSCs secrete pro-inflammatory cytokines, growth factors, and proteases that create a feedback loop, perpetuating the inflammatory state and further impairing HSC function (255). This senescence-associated secretory phenotype contributes to the chronic inflammatory environment in the BM niche, exacerbating the decline in HSC function and promoting the development of hematopoietic disorders.

On the other hand, transient immune challenges can imprint long-term functional modifications. Acute LPS exposure triggers rapid, temporary changes in HSC proliferation and gene expression, which normalize within days (110). However, LPS also induces persistent alterations in chromatin accessibility, specifically in myeloid lineage enhancers, enhancing the responsiveness of genes involved in immunity. The transcription factor C/EBPβ is crucial for maintaining these LPS-induced epigenetic marks and gene expression changes. The deletion of C/EBPβ eliminated the long-term epigenetic marks and gene expression changes induced by LPS, highlighting its role as a pioneer factor that initiates and sustains chromatin accessibility. Direct TLR4 signaling in HSCs is essential for these modifications, as TLR4-deficient HSCs show significantly reduced chromatin changes and responsiveness. This strategic adaptation enables a rapid and robust immune response upon reinfection. In another study, findings reveal that such impairments can be irreversible, with no recovery observed even after a year (108). Exposure to inflammation or bacterial infection leads to a permanent depletion of functional HSCs, resulting in accelerated aging and the emergence of blood and BM phenotypes typically seen in elderly humans but not in aged laboratory mice. During and after inflammatory challenges, HSCs fail to undergo self-renewal, highlighting a cumulative inhibitory effect of discrete inflammatory events over time. This research positions early and mid-life inflammation as a key mediator of lifelong defects in tissue maintenance and regeneration, significantly impacting HSC functionality and potentially leading to age-associated hematologic diseases. Early inflammatory responses inscribe a lasting memory in HSPCs, which can enhance the host’s innate immunity against future infections.

MSC

MSCs are profoundly influenced by inflammatory environments, leading to significant epigenetic modifications that impact their functionality and therapeutic potential. Inflammatory cytokines, such as TNF-α and IL-1β, can induce changes in DNA methylation and histone modification, thereby altering the expression of genes involved in MSC differentiation, proliferation, and migration. These epigenetic changes can enhance or impair the regenerative capacity of MSCs. For instance, inflammation-induced hypomethylation can up-regulate genes that enhance MSC migration and immunosuppression, whereas hypermethylation may silence genes crucial for tissue repair. hMSCs conditioned with Skov-3 medium for 16 d exhibited high levels of tumor associated fibroblasts markers Tn-C, TSP, and FSP and increased expression of a-SMA, FAP, and desmin, which are associated with cell migration and tissue remodeling (111). The same study shows that MSCs contribute to the tumor microenvironment by secretive immunosuppressive and tumor-promoting factors, HFG, EGF, and IL-6, indicating that inflammation-induced hypomethylation can activate genes that support MSC migration and enhance immunosuppressive functions, aiding in tumor progression. Alternatively, the presence of mice MSCs in tumor microenvironment was associated with enhanced fibrotic response, suggesting hypermethylation of genes that are crucial for normal repair, leading to fibrosis as opposed to normal healing (109).

In addition to DNA methylation, histone modifications also play a significant role in the epigenetic regulation of MSCs under inflammatory conditions. In MDS, MSCs in the BM are reprogrammed to support disease progression. In these MDS-MSCs, histone variant isoform mH2A1.1 up-regulation drives pro-inflammatory signaling via the TLR4 pathway, inducing a senescence-associated secretory phenotype and alters the proteomic and metabolic profiles of MSCs (256) (Fig 3C). Further proteomic analysis has shown up-regulation of several proteins, including AHCY and CBX3/HP1γ. Overexpression of AHCY, a protein part of the DNMT1 interactome, leads to genome hypermethylation, whereas CBX3/HP1γ contributes to increased levels of H3K9me3.

Increased H3K9me3 in aged MSCs is associated with several detrimental effects on their functionality and regenerative capacity. Removing KDM4B leads to increased MSC aging and senescence, indicating that higher H3K9me3 levels contribute to this. Losing KDM4B significantly raises the levels of H3K9me3 and HP1α clusters as MSCs age, promoting a more repressive chromatin state (257). Elevated H3K9me3 also significantly increases the nuclear size of MSCs after multiple cell divisions and leads to higher expression of aging-related genes such as Cdkn2a (p16) and Cdkn1a (p21). Overall, the accumulation of H3K9me3 in aged MSCs is linked to increased cellular senescence, reduced self-renewal capacity, and the formation of senescence-associated heterochromatin foci, contributing to the decline in their regenerative potential and functionality.

During aging, MSCs experience significant epigenetic changes driven by chronic inflammation, exacerbating cellular senescence. Inflammatory cytokines such as TNF-α and IL-6 activate the NF-κB pathway, perpetuating a pro-inflammatory environment through ROS (125). This chronic inflammation leads to DNA methylation changes in genes regulating the cell cycle, DNA repair, and differentiation. For instance, hypermethylation of the Dlx5 impairs MSC differentiation. Senescent MSCs show altered gene expression, including miRNAs, resulting in reduced proliferation and structural changes. Oxidative stress further drives these epigenetic alterations, increasing susceptibility to malignant transformation.

ISC

ISCs play a crucial role in maintaining the homeostasis and regeneration of the intestinal epithelium, but their function and regulation can be significantly altered by inflammatory processes, particularly through epigenetic modifications. Key proteins such as IFN-γ and STAT1 are pivotal in mediating these effects. IFN-γ-STAT1 signaling pathway becomes hyperactive during inflammation, leading to up-regulation of MHC-II in aged ISCs (258). The heightened STAT1 transcription factor signaling leads to increased surface expression of MHC-II in aged ISCs. This interaction facilitates CD4⁺ T-cell engagement, perpetuating a cycle of inflammation and immune response.

Epigenetically, inflammation induces significant changes in chromatin accessibility within ISCs. These modifications were examined using single-cell RNA sequencing and gene set enrichment analysis to identify differentially expressed genes and altered pathways. A study revealed that aged ISCs exhibit specific changes in chromatin that remain stable even when cultured in organoids ex vivo (258). These changes include increased accessibility at inflammation-associated loci, which promote a state of sustained inflammation within the epithelium. The differential gene expression analysis identified significant DEGs across various cell types in the intestinal epithelium, indicating that inflammation affects not only ISCs but also TA cells and enterocyte progenitors.

Inflammation triggers a complex array of epigenetic modifications in ISCs, influencing their function and regenerative capacity. Key proteins such as Hopx and Atoh1 are involved in these modifications, playing significant roles in the regulation of ISC differentiation and maintenance (142, 259). During chronic inflammation, such as in inflammatory bowel disease, ISCs are exposed to persistent inflammatory signals that can lead to lasting changes in their gene expression profiles. Inflammatory cytokines, such as IL-11 and type I interferons, have been shown to influence ISC behavior. IL-11, for example, is necessary for mucosal regeneration, whereas type I interferons can impair recovery and reduce ISC proliferation. These inflammatory signals can induce epigenetic modifications, such as DNA methylation, histone modifications, and changes in chromatin structure. For instance, prolonged ER stress and hypoxia, common in chronic inflammation, activate the unfolded protein response, which is crucial for ISC proliferation and differentiation. Studies have shown that HNF4α, a transcription factor involved in ER stress response, is essential for intestinal epithelial repair, linking inflammation-induced ER stress to epigenetic regulation of ISC function (260, 261).

Epigenetic regulation because of inflammation can result in the activation or repression of specific genes that are critical for ISC function. For example, studies using ChIP have demonstrated that inflammatory cytokines can lead to increased histone acetylation at loci associated with pro-inflammatory genes, enhancing their expression. Conversely, DNA methylation analysis has shown that inflammation can cause hypermethylation of genes involved in maintaining stem cell pluripotency, thereby reducing their expression and impairing the regenerative capacity of ISCs. In addition, single-cell RNA sequencing of ISCs isolated from inflamed tissues has revealed altered expression patterns of key regulatory genes, highlighting the impact of chronic inflammation on the epigenetic landscape of these cells.

Recent evidence suggests that not only TA cells but also fully differentiated intestinal epithelial cells possess the potential to de-differentiate and replenish the damaged ISC niche. This raises critical questions about the inherent fate of these cells and the epigenetic mechanisms underlying their differentiation. Signaling molecules such as Wnt, Bmp, and Notch, produced by Paneth cells and myofibroblasts, play a pivotal role in regulating ISC fate and function (260). These pathways interact with the chromatin landscape, which, when in a permissive state, facilitates the binding of lineage-defining transcription factors such as Atoh1 (262). This chromatin accessibility allows for dynamic responses to injury, enabling differentiated cells to revert to a more primitive state and contribute to epithelial repair. Such plasticity is critical in maintaining intestinal homeostasis, particularly in response to acute injuries where the rapid regeneration of the epithelial barrier is essential.

Epigenetic modifications, particularly those involving chromatin structure, are central to the regulation of ISC plasticity. Histone variants, such as H2A.Z, play a significant role in remodeling chromatin and altering gene expression (263). The incorporation of H2A.Z into specific genomic regions, mediated by components such as Znhit1, influences the expression of key genes such as Lgr5 and TGF-β, which are crucial for ISC self-renewal and differentiation (264) (Fig 4C). Deleting ZNHIT1 impacts ISC differentiation directly, without altering Wnt signaling, underscoring its specific role in regulating chromatin dynamics and gene expression in ISCs. Moreover, the transcriptional co-repressors MTG8 and MTG16, and factors such as Id3, modulate chromatin accessibility, maintaining ISC identity and controlling differentiation into secretory lineages (265). The interplay between these epigenetic regulators and transcription factors ensures a balanced differentiation process, allowing for efficient repair and regeneration of the intestinal epithelium.

Neural stem cell (NSC)

NSCs play a pivotal role in neurogenesis, essential for maintaining brain plasticity throughout life. Neuroinflammation in the central nervous system is unique because of the blood-brain barrier, separating the CNS from the peripheral immune system when still allowing an immunological response. Microglia, the resident immune cells in the CNS, are pivotal in initiating innate immune responses through pattern recognition receptors that detect danger signals from dying cells and microorganisms. Upon activation, microglia undergo morphological changes and release nitric oxide, ROS, and various pro-inflammatory cytokines and chemokines, orchestrating a robust M1 response. This M1 response, can lead to chronic inflammation if not properly regulated, with potential long-term consequences for surrounding neural cells (266). Conversely, M2 microglia release anti-inflammatory cytokines such as TGF-β and IL-10, promoting neuroprotection and tissue repair, although the M2 response is often transient in various CNS injury models.

LPS, commonly used to model inflammation in vitro, have been shown to adversely affect NSC viability and differentiation. In LPS-treated NSCs, key epigenetic changes occur, especially involving histone modifications. For instance, prolonged LPS exposure increases the expression of Jmjd2b, a histone demethylase, and NF-κB p65, indicating their roles in inflammation-induced epigenetic regulation (267) (Fig 5). Jmjd2b targets the trimethylated lysine on H3K9me3, a mark typically associated with repressed chromatin, and demethylates it to maintain an open chromatin state conducive to gene transcription. This epigenetic alteration was confirmed through expression analysis and ChIP assays, which revealed reduced H3K9me3 levels at promoters of genes such as Notch1 and IL-1β in long-term LPS-treated cells. The knockdown of Jmjd2b led to decreased expression of various genes, including p65, iNOS, Bcl2, and TGF-β, highlighting its regulatory influence. Pathway analyses further identified down-regulated genes such as NF-κB, Sema3e, Wnt, and Lef1, involved in neurogenesis and cellular differentiation processes. Thus, inflammation through LPS-induced pathways profoundly alters the epigenetic landscape of NSCs, primarily via histone demethylation, which affects key genes and pathways essential for NSC function and neurogenesis. Understanding these mechanisms provides crucial insights into the interplay between inflammation and NSC regulation (268).

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Figure 5. A schematic illustration of epigenetic changes in NSCs because of stress.

LPS adversely affects NSC viability and differentiation by inducing key epigenetic changes, particularly histone modifications, with prolonged LPS exposure increasing Jmjd2b and NF-κB p65 expression, leading to reduced H3K9me3 levels at gene promoters essential for NSC function and neurogenesis, thus profoundly altering the epigenetic landscape of NSCs.

Alternatively, inflammation or injury can cause astrocytes to become reactive, reacquiring characteristics such as neural progenitor cells (NPCs), with pro-inflammatory cytokines such as TNF-α playing a key role in this reprogramming. Research exploring the transcriptome and epigenetic profiles of astrocyte populations has revealed that changes in epigenetic markers, such as histone modifications associated with active promoters, such as H3K4me3 (117), and with repressed promoters, such as H3K27me3 (118), accompany shifts in cellular potential (268). Experimental studies using the mouse NPC line CTX12 have demonstrated that astrocytes’ plasticity and capacity for dedifferentiation are influenced by their differentiation conditions. This understanding of astrocytes’ latent neurogenic potential and the epigenetic mechanisms regulating it is vital for developing regenerative medicine strategies. Notably, reactive astrocytes, naturally present at injury sites, offer a promising target for brain repair mechanisms, underscoring the role of inflammation in activating pathways that reprogram astrocytes to a more plastic, NPC-like state.

MuSC

Aging-related systemic inflammation exerts profound effects on MuSCs, driving substantial alterations in their epigenetic landscape and contributing to their functional decline. Inflammatory signals, particularly those mediated through CCR2 ligands such as CCL2, CCL7, and CCL8, play a pivotal role in this process. These signals elevate the levels of CCR2 chemokines, leading to increased CCr2 expression in myogenic progenitors. This heightened CCR2 activity inhibits MP fusion into myofibers by activating MAPKp38δ/γ signaling and phosphorylating MyoD, which suppresses myogenic commitment factor Myogenin. As a result, the regenerative capacity of muscle is impaired, as myogenic progenitors are less able to form the multinucleated myotubes needed for effective muscle repair exacerbating the decline in muscle function seen with aging (269).

Chronic exposure to these inflammatory signals leads to a significant reduction in the activity of Kmt5a, an enzyme crucial for the monomethylation H4K20. The diminished activity of Kmt5a and the resultant decline in H4K20me1 disrupt MuSC quiescence by down-regulating Notch target genes, essential for maintaining stem cell dormancy. This disruption is exacerbated in aged MuSCs, which exhibit lower levels of both Kmt5a and H4K20me1, creating an epigenetic environment that predisposes them to premature activation and subsequent cell death via ferroptosis (270 Preprint). Reduced Kmt5a activity leads to decreased levels of H4K20me1, a histone modification critical for the formation of constitutive heterochromatin and proper cell-cycle progression. This reduction disrupts the transcriptional programs necessary for MuSC quiescence and survival. Specifically, the loss of H4K20me1 results in the repression of Notch signaling components, including Notch, Jag1, Numb, and Rbpj, through increased promoter-proximal pausing, leading to decreased expression of quiescence-associated genes, such as Hes1 and Hey1, and increased expression of activation-associated genes, such as Myf5 and Dek. Furthermore, the persistent reduction in H4K20me1 prime MuSCs for ferroptosis, characterized by abnormal iron metabolism, elevated intracellular iron levels, increased ROS, and lipid peroxidation. This epigenetic reprogramming, driven by inflammation, sensitizes MuSCs to inflammatory signals, leading to their premature exit from quiescence, susceptibility to ferroptosis, and impaired regenerative capacity.

In young muscle, injury induces a coordinated inflammatory response, starting with the recruitment of neutrophils, eosinophils, and macrophages. This triggers a pro-inflammatory phase that promotes myogenic proliferation through cytokines such as IL-6 and TNF-α (113, 271) followed by an anti-inflammatory phase aiding differentiation and repair via TGF-β and IGF-1 (272, 273). However, in aged muscle this becomes chronic, leading to persistent inflammation that hampers regeneration (274) (Fig 6C). For example, increased IL-6 signaling in aged mice causes MuSC exhaustion and atrophy. Epigenetically, chronic inflammation disrupts p38α/β MAPK, linked to PRC2, adding repressive H3K27me3 marks on myogenic gene promoters such as Pax7 (247). After activation by TNF-α, the p38α kinase phosphorylates EZH2 which enhances the binding affinity of EZH2 to transcription factor YY1. The p38α, YY1, and EZH2 complex is recruited to the Pax7 promoter, catalyzing the trimethylation of H3K27. PAX7 directly influences myogenic gene expression by inducing chromatin modifications through histone methyltransferase complexes (275). Notably, PAX7 up-regulates Myf5, a key regulator of muscle commitment, via association with the Wdr5-Ash2L-MLL2 HMT complex, which methylates histone H3K4. This marks chromatin as active, facilitating the activation of MYF5. ChIP studies confirmed that PAX7 recruits this complex to gene promoters, promoting H3K4 methylation and ensures transcription of myogenic genes. In addition, PAX7’s association with demethylated and trimethylated H3K4 regions shows its role in maintaining active chromatin states.

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Figure 6. A schematic illustration of epigenetic changes in MuSCs because of various stressors.

(A) Bmi1 is shown to maintain MuSC pools and myogenesis during muscle via repression of the ink4a locus after muscle injury and during oxidative stress. Bmi1 overexpression in mouse models and patients enhances MT1-mediated protection against oxidative stress, improving muscle strength and regeneration, likely through gene silencing mechanisms involving PRC1. (B) PRMT5 represses p21 expression via H3R8 dimethylation at regulatory sites upstream of the murine p21 gene. PRMT7 regulates p21 expression through DNMT3b-mediated hypermethylation at the p21 promoter, influencing H4R3me2 and H3K4me3 levels. (C) Inflammatory signals, especially CCR2 ligands, elevate CCR2 expression in myogenic progenitors, inhibiting their fusion into myofibers by activating MAPKp38δ/γ signaling and phosphorylating MyoD. Chronic inflammatory signals reduce Kmt5a activity, leading to a decline in H4K20me1 levels, disrupting MuSC quiescence by down-regulating Notch target genes. In addition, chronic inflammation disrupts p38α/β MAPK, adding repressive H3K27me3 marks on myogenic gene promoters such as Pax7.

In pathological conditions such as muscular dystrophies, persistent inflammation drives extensive epigenetic reprogramming. Chronic inflammation in these diseases elevates levels of cytokines that continuously activate pathways leading to both DNA methylation and histone modifications. This reprogramming not only suppresses myogenic differentiation but also enhances fibrogenic pathways, shifting the balance towards fibrosis and muscle degeneration (276).