Epigenetic regulation of adult stem cell function.

REVIEW ARTICLE

Epigenetic regulation of adult stem cell function Lorenzo Rinaldi1,2,3 and Salvador Aznar Benitah3,4 1 2 3 4

Centre for Genomic Regulation, Barcelona, Spain Universitat Pompeu Fabra, Barcelona, Spain Institute for Research in Biomedicine, Barcelona, Spain Catalan Institution for Research and Advanced Studies, Barcelona, Spain

Keywords adult stem cells, ageing and cancer, epigenetic regulators, lineage commitment, tissue homeostasis Correspondence S. A. Benitah, Institute for Research in Biomedicine (IRB Barcelona), C/ Baldiri Reixac 10, 08028 Barcelona, Spain Fax: 0034 93 403 71 14 Tel: 0034 93 403 40 21 E-mail: [email protected] (Received 12 June 2014, revised 17 July 2014, accepted 22 July 2014) doi:10.1111/febs.12946

Understanding the cellular and molecular mechanisms that specify cell lineages throughout development, and that maintain tissue homeostasis during adulthood, is paramount towards our understanding of why we age or develop pathologies such as cancer. Epigenetic mechanisms ensure that genetically identical cells acquire different fates during embryonic development and are therefore essential for the proper progression of development. How they do so is still a matter of intense investigation, but there is sufficient evidence indicating that they act in a concerted manner with inductive signals and tissue-specific transcription factors to promote and stabilize fate changes along the three germ layers during development. In consequence, it is generally hypothesized that epigenetic mechanisms are also required for the continuous maintenance of cell fate during adulthood. However, in vivo models in which different epigenetic factors have been depleted in different tissues do not show overt changes in cell lineage, thus not strongly supporting this view. Instead, the function of some of these factors appears to be primarily associated with tissue functionality, and a strong causal relationship has been established between their misregulation and a diseased state. In this review, we summarize our current knowledge of the role of epigenetic factors in adult stem cell function and tissue homeostasis.

Introduction The role of epigenetic mechanisms in establishing different cell fates during embryogenesis is fairly well understood. However, if and how epigenetic mechanisms are necessary to stably maintain the identity of adult stem cells and their progeny during tissue homeostasis and repair is still under intense investigation. In recent years, a considerable number of studies have shown that factors involved in chromatin regulation during embryogenesis have diverse roles in adulthood, which are not always related to epigenetics.

Adult stem cells maintain adult tissue homeostasis by replenishing dying or damaged cells or by regenerating injuries. However, since tissues significantly differ in their turnover rates, each imposes a different demand for cellular input from its resident stem cells. Intriguingly, a variety of mechanisms have evolved in different adult stem cells that involve the interplay between quiescent and actively proliferating stem cell populations, stochastic regulation of symmetric or asymmetric stem cell divisions, neutral competition of the stem cell progeny, or combinations of these, all of which effi-

Abbreviations 5-Mc, 5-methylcytosine; DMR, differentially methylated regions; DNMT, DNA methyltransferase; epSC, epidermal stem cell; ESC, embryonic stem cell; HSC, hematopoietic stem cell; NSC, neural stem cell; PcG, polycomb group; PRC, polycomb repressive complex.

FEBS Journal (2014) ª 2014 FEBS

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Epigenetic regulation of adult stem cell function

L. Rinaldi and S. A. Benitah

ciently ensure tissue homeostasis [1,2]. In addition, some tissues, such as the keratinocyte compartment of the skin, the mammary gland or the prostate, are highly compartmentalized and rely on distinct populations of stem cells, often located adjacent to each other, that display completely different behaviours [1,3–7]. How different stem cells are stably established during embryogenesis, and how they retain or lose their identity while self-renewing or differentiating respectively during steady state, is still largely unknown. Epigenetic modifications are chemical modifications either of double strand DNA or of the core nucleosome histones, catalysed by different families of enzymes. These modifications alter the structure of chromatin by either facilitating or preventing the binding of transcription factors and other cofactors. Importantly, although the turnover of some of these modifications is far more rapid than previously thought, often they are stably transmitted after a cellular division and can therefore affect the identity of the progeny. In this sense, epigenetic mechanisms allow genetically identical cells to stably adopt different

phenotypes by controlling the transcription availability of different parts of the genome packaging or opening different parts of the chromatin [8,9]. In fact, findings from many laboratories in recent years indicate that the combination of transcription factors and chromatin remodelling factors might be essential for different aspects of the biology of adult stem cells. Importantly, a number of epigenetic factors are mutated in human cancers or deregulated during ageing, suggesting that perturbations in their functions may be causative of disease. This review focuses on recent advances in our understanding of the roles of different epigenetic mechanisms in regulating adult stem cell specification and function during homeostasis (Table 1).

Modification of chromatin and DNA Histone modifications Histones can be post-translationally modified at different amino acid residues through methylation, acetylation, ubiquitination, sumoylation and phosphorylation.

Table 1. Summary of the role of epigenetic factors in different adult stem cells and phenotypes associated with their genetic manipulation in vivo. Main epigenetic complexes PRC1

PRC2

5Mc/DNMTs

5-Hmc/TET G9A/GLP

MLL1/3

2

Function in adult stem cells

Epigenetic mechanism disruption

Reference

Self-renewal of hematopoietic, epidermal and neural stem cells Regulation of cell cycle and inhibition of senescence and differentiation

Defects in blood cell maturation

[50–57,81,82,121,122]

Hematopoietic stem cell self-renewal and differentiation Differentiation of neural and epidermal stem cells Maintenance of hematopoietic stem cell, epidermal stem cell, neural stem cell and muscle stem cell (satellite cell) self-renewal Drives hematopoietic stem cell differentiation upon transplantation (Dnmt3a) Abundant in neural stem cells Drives expression of neuronal genes Hematopoietic stem cell differentiation Specification of neurons during development and under external stress Represses adipogenesis Hematopoietic stem cell self-renewal Promotes adipogenesis

Cell cycle defects and premature differentiation of epidermal stem cells Neurological defects Reduced blood and neural stem cell activity

[58–61,76–79,119,120]

Hair follicle cycling defects, and epidermal differentiation Impaired number of mature blood cells

[62–67,96,97,105–107]

Loss of Dnmt1 results in spontaneous and irreversible differentiation of human epidermal stem cells Impaired neural activity causing cognitive defects Impairment in spatial learning and memory Reduced number of neural stem cells Delayed blood stem cell differentiation Altered neuronal gene expression causing impaired cognitive functions

[111–114]

Increased fat accumulation and tissue weight Loss of hematopoietic stem cell self-renewal potential Reduction of fat accumulation

[71–126]

[69–99,125]




The function and activity of these proteins has been extensively reviewed elsewhere [8–12]. In this section we will briefly introduce just those epigenetic factors whose activity has been shown so far to affect the biology of adult tissues. Amongst the most studied histone modifiers are polycomb group (PcG) proteins. PcG proteins were identified in Drosophila and have since been shown to be essential for early development, X chromosome inactivation, adult homeostasis and reprogramming to pluripotency [10,12–15]. PcG proteins promote chromatin remodelling and compaction that leads to a transcription inhibition mainly through the formation of polycomb repressive complexes (PRCs) [9,11]. The two major PRCs, named PRC1 and PRC2, mediate the ubiquitination of histone H2A and the methylation of histone H3, respectively. The core PRC2 is formed by the subunits EED, Suz12 and the histone methyltransferases Ezh1 and Ezh2. On the other hand, Ring1a/b and members of the Pcgf and PhC family of proteins form the PRC1 complex. The classical model of PRC2–PRC1 action establishes that PRC2 methylates lysine 27 of histone H3 (H3K27me3) and that the PRC1 complex recognizes this modification through the chromodomain of its Cbx subunit. PRC1 in turn ubiquitinates lysine 119 at histone H2A (H2AK119ub) through the ring finger protein RING1A/B (Fig. 1A). However, recent studies indicate that this linear model is not always followed, and that the composition of PRC2 and PRC1 varies according to different possible combinations of their subunits (i.e. whether PRC2 contains Ezh2 or Ezh1, or whether PRC1 contains the proteins Bmi1 or Mel18) and is context dependent (Fig. 1B) [16]. In this sense, PRC1 complexes that do not even contain a Cbx subunit capable of interacting with H3K27me3 have been

identified and shown to play an important role during early embryogenesis [10,17–20]. Furthermore, although PRC2 was originally identified as a repressor associated with the presence of H3K27me3 mark, evidence suggests that EZH1 can locate to actively transcribed genes independently of its histone methyltransferase activity [21]. This interesting complexity in the composition and function of PRC2 and PRC1 might in fact endow them with the ability to contribute to different aspects of embryonic development and adult tissue maintenance in a context-dependent manner. Deletion of essential members of both complexes is incompatible with a proper development, underscoring their importance in early embryogenesis [9]. PcG proteins also interact with other epigenetic factors such as G9a and members of the thritorax family proteins. G9a is responsible for establishing dimethylation of lysine in position 9 on histone 3 (H3K9me2), a mark that is often associated with transcriptional repression. Interestingly, H3K9me2-rich regions are organized in ‘large organized chromatin K9 modification blocks’ (LOCKs), that are highly conserved between mammals and have been proposed to play a crucial role compacting chromatin during the commitment of embryonic cells into diverse somatic lineages such as neurons and hepatocytes [22]. Similar to PRC1 and PRC2, the activity of G9a is indispensable for proper development, and its deletion in embryonic stem cells (ESCs) results in embryonic lethality [23]. Another family of epigenetic factors, the thritorax group proteins, is involved in gene activation and they both interact with PcG proteins and other histone modifications. Thritorax complex allows gene expression by producing the trimethylation of lysine 4 at histone 3 (H3K4me3) [24]. H3K4me3 is well established to be

A

Fig. 1. Schematic representation of the regulation of histone post-translational modifications by PRC1 and PRC2 complexes. (A) PRC2 complex deposits H3K27me3, which is recognized by PRC1 containing a Cbx unit. PRC1 in turn catalyzes the ubiquitination of H2A lys 119, resulting in transcriptional repression. (B) A non-canonical interplay between PRC1 and PRC2 also leads to gene repression, whereby PRC1 can establish H2Aub119, which serves as a docking site for PRC2 and subsequent H3K27 trimethylation.


B

3


present at the promoter of active genes. The first and most described thritorax member is MLL1, initially noted for its high frequency mutation rate in human cancers. Other MLL proteins are involved in methylating H3K4 at gene regulatory regions such as enhancers, suggesting that their role might also be important for fate decisions during embryogenesis [24–26]. Modifications of double strand DNA DNA methylation occurs on the fifth position of the cytosine (5-Mc) at the dinucleotide cytosine-guanine (CpG), and it is involved in the regulation of several aspects of gene expression such as long-term gene silencing, transcriptional elongation and maintenance of genomic stability. The mechanisms underlying the establishment, maintenance and removal of DNA methylation have been extensively reviewed elsewhere [27,28]. Briefly, during mammalian development, the pattern of DNA methylation is established by the de novo DNA methyltransferases DNMT3A and DNMT3B [29–31]. This pattern of DNA methylation is in turn primarily maintained by DNMT1, although with the help of DNMT3A and DNMT3B. In addition, an accessory inactive DNA methyltransferase (DNMT3L) modulates their activities [32] (Fig. 2). DNA methylation, mainly if clustered, has always been associated with gene repression; however, in the last few years, genome-wide methylation analyses have revealed a large variety of patterns of DNA methylation that do not always correlate with the regulation of gene expression. Generally, however, if the promoter of a gene is methylated, it will not be actively transcribed. Conversely, if the exons of the gene have high levels of 5-Mc, this correlates with an actively transcribed gene. Finally, regions of compacted chromatin such as heterochromatin sites, retrotransposons, microsatellites and telomeres normally have very high levels of methylation [33–35]. However, it is still unclear whether changes in DNA methylation are


causative of regulating gene expression per se or rather important for the long-term stabilization of the threedimensional conformation and/or gene expression activity downstream of other regulatory proteins. Recent studies in adult stem cells support this last hypothesis [36,37]. Interestingly, 5-Mc is a reversible modification, since methylated cytosines can be either actively or passively demethylated [27,28]. Passive demethylation occurs during cell replication when DNA methyltransferases might be inhibited or absent, resulting in a locus-specific or global loss of DNA methylation [28,38,39]. On the other hand, active demethylation relies on the ability of 5-Mc to be oxidized into 5-hydroxymethylcytosine (5Hmc) by TET proteins [40] (Fig. 2). For instance, in the very early stages of the formation of the mammalian embryo, DNA is highly demethylated by TET1/2 with a progressive and transient increase of hydroxymethylation. Subsequently, the de novo DNMTs re-establish the correct pattern of DNA methylation in the second week of the murine embryonic stage. Interestingly, DNA methylation does not appear to occur independently of histone modifications, and their concerted action might be essential for the correct spatial and temporal regulation of cell fates during development [9,10,13,30,31,41,42]. How DNA methylation and hydroxymethylation are dynamically regulated during adulthood and their impact in adult tissue homeostasis will be discussed below. Other epigenetic mechanisms Recently, much attention has been paid to studying the role of other epigenetic mechanisms that might regulate gene expression throughout modifications in the high order three-dimensional conformation of chromatin. Amongst these, distant regulatory regions, also called enhancers, have been defined by a combination of distinct histone marks including acetylation of lysine 27 in histone 3 (H3K27ac) and for having a high

Fig. 2. Schematic representation of the role of de novo DNA methyltransferases, maintenance DNA methyltransferases and TET proteins in regulating the methylation and demethylation of DNA.

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ratio of monomethylation at lysine 4 in histone H3 (H3K4me1) versus trimethylation of the same residue (H3K4me3) [43–45]. Enhancers are abundant in the mammalian genome and they normally show low levels of DNA methylation [25,46]. The importance of these distant regulatory regions is still under investigation, but it is thought that enhancers are capable of driving and stabilizing gene expression in different types of cells during embryogenesis, although their role in adult stem cell homeostasis is still unknown [45,47].

Epigenetics of adult stem cells and adult progenitors Hematopoietic stem cells The hematopoietic system has one of the highest turnover rates among adult tissues. Hematopoietic stem cells (HSCs) residing at the bone marrow are primarily responsible for the daily maintenance of hematopoietic homeostasis during adulthood [48]. Two types of HSC states coexist, a quiescent population thought to constitute a long-term reservoir for injury repair, and an actively dividing short-term subset mainly responsible for the daily sustenance of haematopoiesis [48]. These two populations also differ in their long-term selfrenewal potential. HSCs can commit to two branches, the common lymphoid progenitor or the common myeloid progenitors. Lymphoid progenitors differentiate into diverse lymphocytes, mostly B- and T-cells, whereas common myeloid progenitors instead give rise to all the other blood cells, including platelets, erythrocytes and other immune cells [49]. PRC1 and PRC2 in HSC self-renewal and differentiation Several studies have shown that chromatin remodelling factors are important regulators of HSC self-renewal and differentiation. For instance, PRC1 is important for self-renewal of HSCs. In this sense, forced expression of Bmi1 in mouse ESCs leads to an upregulation of genes involved in haematopoiesis [50], and its overexpression in adult HSCs enhances their self-renewal potential. Accordingly, deletion of BMI1 impairs HSC function and results in defects in the bone marrow microenvironment that sustains HSC homeostasis [51]. Furthermore, deletion of other PRC1 members such as MEL18, Phc1, Phc2, RING1A/B and Cbx2 results in a phenotype similar to that of the deletion of BMI1 [9,52]. Mechanistically, PRC1 maintains HSC longterm self-renewal through the repression of the cell cycle inhibitors p16INK4a and p19ARF [53–55]. In FEBS Journal (2014) ª 2014 FEBS


accordance with this, the deletion of the p16ink4a or P19Arf genes partially rescues the defects in HSC BMI1-deficient mice [53]. In addition, BMI1 prevents premature lineage specification of both HSCs and multipotent progenitors, repressing directly lymphoid lineage genes such as EBF1 and PAX5 [54]. However, PRC1 is not only involved in HSC selfrenewal but is also required for efficient HSC differentiation. Five different Cbx subunits can interact with the core PRC1 complex and are capable of recognizing the histone mark H3K27me3 deposited by PRC2 [56]. Two independent studies have recently shown that the expression of Cbx proteins, and their incorporation into the PRC1 complex, is dynamically regulated during the process of ESC differentiation [17,19]. Cbx7 is the main Cbx subunit expressed in pluripotent ESCs and is exclusively associated with the PRC1 to repress the expression of lineage commitment genes and the other Cbx subunits, with the exception of Cbx6 [17]. However, when ESCs differentiate, Cbx7 is replaced by Cbx8, Cbx4, Cbx2, resulting in different PRC1 complexes that delineate the commitment of ESCs into the three germ layers, while repressing genes that promote pluripotency, including Cbx7 [17–19]. Thus, an intriguing PRC1 auto-regulatory loop is required to balance ESC pluripotency and differentiation. Interestingly, this dynamic behaviour is conserved also in HSCs. In long-term HSCs, PRC1 contains predominantly Cbx7, whose activity enhances HSC cell proliferation and self-renewal [57]. Conversely, during the commitment of HSCs into the lymphoid or myeloid lineages, expression of Cbx7 decreases and is replaced by Cbx8, Cbx2 or Cbx4 within PRC1, which in turn induce HSC differentiation [57]. Similar to PRC1, PRC2 might act at different levels along the axis of HSC self-renewal and differentiation. For instance, mutations that inactivate PRC2 components including SUZ12, EZH2 or EED enhance HSC activity. In addition, PRC2-deficient HSCs are more capable of repopulating the hematopoietic system than the wild type [52,58]. However, intriguingly, conditional deletion of EED enhances HSC/progenitor activity, as in null mutation conditions, but renders HSCs incapable of properly differentiating, leading to an exhaustion of the long-term HSC pool [59]. The impaired proliferation of EED-deficient HSCs is due to high expression of the cell cycle genes p16ink4a and p19Arf and can only be partially rescued by the deletion of CDKN2A. However, knockout of CDKN2A cannot rescue the inability of EED-null HSCs to differentiate [59], whereas it restores the hematopoietic defects in BMI1 and EZH1 knockout mice [51,60]. In addition, deletion of either EZH1 or EZH2 in HSCs 5


results in different and weaker HSC functional defects than by the deletion of EED. Altogether, this suggests that different PRC2 complexes formed by distinct combinations of its subunits might impinge on HSC function at different levels. It can also be speculated that these different PRC2 subunits might have PRC2independent functions in haematopoiesis [59]. For instance, EED acts as a linker between PRC2 and PRC1 function in ESCs by helping recruit PRC1 to H3K27me3 and enhancing its ubiquitinase activity [61]. Thus, this dual interactive mechanism of EED with PRC2 and PRC1 could explain why its deletion in HSCs results in such a strong phenotypic effect compared with other PRC2 members. DNA methyltransferases in HSCs: self-renewal or differentiation? DNA methylation has been identified as an epigenetic modification important for several aspects of haematopoiesis. For instance, differential regulation of DNA methylation is important for the specification of HSCs into myeloid versus lymphoid progenitors [62]. However, DNA methylation also maintains HSC multipotency, but might also be required for the stable establishment of all blood lineages derived from common lymphoid and myeloid progenitors [62,63]. Accordingly, HSCs deficient in the maintenance DNA methyltransferase Dnmt1 are incapable of differentiating into all the blood lineages, and interestingly also disrupt the bone marrow HSC niche [64]. The role of the de novo DNA methyltransferases Dnmt3a and Dnmt3b in HSC function is less clear. A report by Tadokoro et al. (2007) indicated that both proteins are required for the long-term maintenance of HSCs but not for their differentiation into the myeloid and lymphoid lineages [65]. However, the opposite role has recently been described for DNMT3A in murine HSCs. Deletion of DNMT3A in HSCs resulted in no overt hematopoietic phenotype in unchallenged conditions. On the other hand, upon serial transplantation to reconstitute the hematopoietic system of lethally irradiated mice, DNMT3A-deficient HSCs progressively accumulated in their multipotent state and displayed a significantly reduced potential to differentiate [66]. Interestingly, work from the same group has recently shown that DNMT3A is responsible for maintaining borders between regions with high and low levels of 5-Mc that regulate the expression of genes essential for HSC self-renewal and differentiation [67]. Furthermore, the DNA methylation of these regions is altered in aged HSCs, as discussed below [68]. Despite these facts, the specific role of Dnmt3a and Dnmt3b in 6


regulating DNA methylation and the precise impact over gene expression of the pattern of DNA methylation they establish towards the function of HSCs and all lineages downstream has not been determined. Function of histone methyltransferases in HSCs As mentioned above, the methyltransferase G9a together with its partner GLP is essential to establish and maintain the repressive mark H3K9me2. The expression of G9a/GLP, and the levels of its functional readout H3K9me2, increase during HSC differentiation [69]. Regions containing high levels of H3K9me2 can be organized into large organized chromatin blocks, known as LOCKs [22]. In this sense, it has been hypothesized that the increased enrichment for H3K9me2-containing LOCKs during HSC differentiation might diminish transcriptional noise and increase the robustness of the activity of transcription factors that drive HSC differentiation [69]. In accordance with this, depleting human HSCs of G9a/GLP delays their capacity to differentiate in vitro [69]. On the other hand, deletion of G9a in murine HSCs does not result in any relevant phenotype but is required for the proliferation of leukaemic cells in an H3K9me2-dependent manner, thereby impairing the progression of leukaemia in vivo [70]. Another histone methyltransferase that is involved in haematopoiesis is MLL, a member of the thritorax complex that catalyses the methylation of lysine 4 at histone 3 (H3K4) [71]. Embryonic deletion of MLL results in reduced numbers of foetal HSCs [72]. The long-term quiescent pool of HSCs is reduced, and their ability to restore a functional hematopoietic system when transplanted into lethally irradiated mice is significantly impaired. Interestingly, deletion of MLL in adult hematopoietic cells does not affect HSC numbers, but these MLL-deficient HSCs remain incapable of regenerating blood cells in irradiated recipients [26]. Intriguingly, this phenotype is not dependent on the ability of MLL to methylate histone H3 on MLL target genes but rather on its interaction with the histone H4K16 methyltransferase MOF, suggesting that the cooperation of two chromatin remodelling factors is essential for HSC activity [72]. Epigenetic factors regulating epidermal stem cell function The keratinocyte compartment of the skin is formed by the epidermis and appendages including hair follicles, sebaceous glands and sweat glands [3,73]. The epidermis and the structures physically linking it to the FEBS Journal (2014) ª 2014 FEBS


hair follicles are under constant demand of cellular replenishment, imposing a strong pressure over its resident stem cells. Conversely, the hair follicles undergo bouts of hair growth followed by long periods of dormancy. Each compartment is maintained by its own subset of stem cells that accordingly show a very different behaviour [3]. So far several populations of stem cells have been identified in all these compartments, although it is not known how epigenetic mechanisms influence their establishment during development [74] or their maintenance in adulthood [3,73]. Recent reports have nevertheless provided much information on how chromatin remodelling factors regulate the behaviour of some of these stem cell subsets during the homeostasis of their particular compartment in adulthood. These will now be discussed. Histone modifications in stem cells of the epidermis and hair follicles The stem cells of the epidermis must cope with a constant demand of cellular supply due to the loss of cornified cells in the uppermost layer of the epidermis. Interestingly, the choice that actively dividing epidermal stem cells (epSC) make is under a stochastic regulation, whereby it cannot be predicted whether a basal cell in the epidermis will divide symmetrically into two stem cells or two differentiated cells or asymmetrically to produce one daughter stem cell and a differentiated cell [1,75]. Whether these stochastic choices require the influence of chromatin remodelling factors to allow for transcriptional noise and plasticity is not known. Furthermore, similar to HSCs, murine and human epidermis also contains a relatively quiescent population of stem cells thought to contribute primarily to damage repair [1,3]. However, once the stochastic fate of the epidermal stem cell has been determined, its quiescence, proliferation or differentiation does require the concerted activity of different epigenetic complexes [74]. For instance, the activity of the PRC2 complex promotes the proliferation and prevents the differentiation of embryonic and new-born mouse epidermis [76,77]. Accordingly, the expression of Ezh2 and Eed is reduced as epSC differentiate, and the removal of the repressive H3K27me3 mark in differentiation genes by the histone demethylase Jmjd3 is necessary for their efficient differentiation [77,78]. Interestingly, the expression of EZH1 follows the opposite pattern from EZH2 upon differentiation, and deletion of EZH2 results in a reduction of H3K27me3 in basal stem cells but not in suprabasal differentiated cells [78]. This suggests that Ezh1 might be required for the deposition of H3K27me3 in genes that should not be expressed in FEBS Journal (2014) ª 2014 FEBS


suprabasal differentiated epidermal cells [78]. In line with this, deletion of EZH1 and EZH2 results in a hyperproliferative epidermis rather than the hypoproliferation observed in single Ezh2 epidermal knockout mice [79]. Similar to the epidermis, the deletion of EZH2 and EZH1 results in massive defects in the production of mature hair follicle structures, blocking hair follicle stem cells in a non-cycling state due to upregulation of the INK4b (p15) locus [79]. Besides PRC2, PRC1 also affects the function of epSC at different levels. For instance, the PRC1 subunit Cbx4 is highly expressed in basal epSC but, contrary to PRC2, prevents their rapid cycling protecting as well as protecting them from differentiation [80]. Cbx4 also protects basal epSC from entering to a premature senescence through the repression of p16 [80]. Mechanistically, Cbx4 relies on its PRC1 activity and also on its E3-SUMO ligase function, which is PRC1 independent, to regulate epidermal stem cell proliferation and differentiation [80]. On the other hand, BMI1 is expressed both in the basal proliferating and suprabasal epidermal cells and is thought to prevent senescence through repression of the p16ink4A locus [81]. It is not clear, however, whether the interplay between Cbx4, Bmi1 and the PRC2 complex is necessary to balance the quiescence and proliferation of epSC or whether they act on different subsets of stem cells. Future in vivo studies combining loss of function and lineage tracing studies will provide insights into these questions. Epigenetic players interact with several transcription factors to maintain stem cell homeostasis. One example in epSC is the transcription factor p63, a master regulator of epidermal stemness, differentiation and stratification that can act both as an activator and a repressor of gene expression [82]. In doing so, p63 regulates cell cycle proteins and the early and terminal epidermal differentiation markers required for proper epidermal homeostasis. The expression of p63 is regulated by the histone methyltransferase Setd8/PR-SET7/ KMT5a, an enzyme required for the monomethylation of lysine 20 at histone H4 [83]. Accordingly, deletion of Setd8 results in a reduced expression of p63 and in a failure to form stratified epidermis [83]. Another epigenetic mechanism related to p63 involves the histone deacetylases 1/2 (HDAC1/2) [84]. HDAC1/2 remove histone acetylation marks, resulting in chromatin compaction and in repression of transcription. Epidermal deletion of HDAC1/2 impairs the ability of p63 to repress the transcription of its target genes, resulting in a dramatic failure of mature hair follicle structures and in an impairment of epidermal stratification [84]. A third epigenetic mechanism required for regulating 7


p63-dependent transcriptional repression in epSC is lymphocyte-specific helicase (LSH) [85]. LSH is a member of the SNF2 chromatin remodelling factor family whose expression is positively regulated by p63, indicating that both proteins act in a positive feedback manner [85]. p63 also utilizes chromatin remodelling factors to promote epidermal stratification. In this sense, the expression of a cluster of genes necessary for the correct cornification of cells (known as the epidermal differentiation cluster) downstream of p63 requires Satb1 and Brg1, two proteins that open and spatially relocate the chromatin of this region within the nucleus, thereby favouring the transcription of the genes located within [86,87]. Besides p63, the transcription factor Myc is also essential for epidermis homeostasis [88]. When activated, Myc induces proliferation and lineage-specific differentiation of epSC. Myc interacts with several epigenetic factors and its activation is followed by changes in the configuration of euchromatin, specifically depending on the presence of H3K4me3 on its target genes [89]. The overexpression of an activated Myc in the epidermis leads to hyperproliferation followed by strong differentiation of epSC [90,91]. Therefore, activation of Myc must be firmly regulated in order to avoid premature differentiation of epSC [88]. One of the key players of this mechanism is the HDAC Sin3a that not only antagonizes Myc activity by repressing the expression of Myc target genes, but also deacetylates activated Myc to induce its degradation [92]. This explains why deletion of Sin3a in mouse epidermis results in a hyperproliferation and in premature differentiation of epidermal stem cells [92–95]. DNA methylation in epSC The expression of the maintenance DNA methyltransferase DNMT1 is high in the basal layer of the epidermis, and decreases as the epidermis stratifies. DNA methylation follows the same pattern of DNMT1 expression, and analysis of DNA methylation content by MeDIP-ChIP revealed that 5-Mc decreases its amplitude in differentiated keratinocytes compared with epidermal basal cells [96]. Interestingly, DNMT1deficient human epSC cannot fully maintain their pattern of DNA methylation and undergo premature and irreversible differentiation [96], although it is not known to what extent this effect is entirely dependent on the observed changes in DNA methylation. Intriguingly, conditional deletion of DNMT1 in mouse epidermis does not affect the development of the epidermis or its appendages, and only shows signs of uneven epidermal thickness and a reduction of the 8


length of the hair shafts upon ageing [97]. In accordance with this, genome-wide single base pair resolution analysis revealed very similar patterns of DNA methylation between quiescent and active proliferating hair follicle stem cells isolated from young mice [36]. Nevertheless, this study reports more than 2000 differentially methylated regions (DMRs), although these regions were not correlated with changes in gene expression. Considering that deletion of DNMT1 results in hair follicle defects only upon ageing, it will be of interest to study whether changes in DNA methylation become prominent as hair follicle stem cells age, similarly to what has been observed in HSCs. Epigenetics in neural stem cells Neural stem cells (NSCs) constitute a heterogeneous population of adult stem cells that differentiate into mature neurons through a slow maturation process that can last several months until they are all fully connected to local nervous circuits of the central nervous system [7]. As for HSCs, NSCs are nested in specific niches, mainly the subventricular zone of the brain, where they interact with niche components that profoundly affect their behaviour [7]. Several studies have described a fundamental role for epigenetic factors during neurogenesis. We shall now review how epigenetics mechanisms regulate neurogenesis and how they regulate NSC behaviour in the adult brain. Histone modifications in neurons and NSCs Disruption of the histone H3K9 dimethyltransferases G9a and GLP in postnatal neurons alters the transcription of neuronal genes in a way that does not affect neuronal architecture and morphology but results in an impairment of the cognitive functions of the brain and its ability to perceive external stimuli [98]. Interestingly, G9a is also important for the specification of neurons during development and under environmental stress, indicating that it can exert different functions in the brain that are context dependent [99]. Other epigenetic factors play important roles in neural homeostasis. For instance, different members of the PcG proteins are necessary for the in vitro specification of ESCs into neural progenitors [14]. PcG proteins are also responsible for regulating adult NSC self-renewal, or the balance between neurogenesis and gliogenesis. BMI1, as shown in other adult stem cell populations, promotes self-renewal of NSCs by repressing the cell cycle inhibitors p16INK4a and p19ARF [100]. Accordingly, deletion of BMI1 in NSCs reduces their self-renewal potential leading to neurological defects FEBS Journal (2014) ª 2014 FEBS


and cognitive impairment [101]. On the other hand, loss of the PRC2 subunit EZH2 in cortical progenitors results in the upregulation of differentiation genes that are normally decorated by H3K27me3 in these cells, leading to a skewing from self-renewal to differentiation in the cerebral cortex [102]. EZH2 also controls the rate of the cell cycle in NSCs by repressing neuronal differentiation genes, and its deletion impairs neurogenesis while favouring neuronal differentiation [103].


pattern of DNA methylation of neurogenesis genes. Functionally, the decrease in the number of NSCs in TET1-deficient mice results in an impairment in spatial learning and memory cognition [115]. These studies therefore reveal that perturbing the machinery that regulates DNA methylation and hydroxymethylation profoundly affects neurogenesis and NSC function. However, future studies will be required to determine whether a direct causal link between DNA methylation and hydroxymethylation with these observed phenotypes exists.

DNA methylation in NSC self-renewal and differentiation

Epigenetic factors in muscle stem cells

The machinery responsible for establishing DNA methylation also plays essential roles during neurogenesis that include the regulation of the cell cycle, cell death and dendritic growth of NSCs. All three DNA methyltransferases DNMT1, DNMT3A and DNMT3B are expressed in neuronal progenitors and play important roles during neurogenesis [104]. Deletion of DNMT1 in NSCs decreases both neural progenitors and mature neurons caused by an aberrant upregulation of astrocyte-specific genes and genes involved in inflammation [105]. DNMT3A exerts a similar role maintaining NSC identity by repressing the expression of glial differentiation genes and enhancing neurogenic factors [106]. Accordingly, its deletion results in a significant reduction in the number of mature neurons. Interestingly, the genes downregulated in DNMT3A-deficient NSCs were enriched for H3K27me3 confirming an antagonism between DNA methylation and polycomb repressive mechanisms in NSCs [106]. Unexpectedly, deletion of DNMT1 and DNMT3A from adult forebrain neurons does not cause any observable effect [107]. However, the deletion of both DNA methylases from adult neurons impairs cognitive functions such as learning and memory caused by an impaired neuronal activity in the hippocampal regions [107]. Although the precise role of DNMT3B in adult neurogenesis is not known, mutation of DNMT3B is linked to the development of immunodeficiency centromere instability syndrome in humans [108–110]. Besides DNA methylation, DNA hydroxymethylation is abundant in the central nervous system compared with all other adult tissues [111–113]. At the genomic level, 5hmC is enriched at gene bodies of neuronal genes and it increases during neuronal differentiation [114]. TET family enzymes catalyse 5-hydroxymethylcytosine (5-Hmc), and deletion of TET1 decreases the number of adult NSCs and their selfrenewal potential, perhaps by producing an abnormal

The satellite cells are quiescent adult stem cells that upon damage become active and generate new muscle fibres while self-renewing [116–118]. Similarly to other tissues, PcG complexes have important roles in regulating myogenesis. EZH2 (PRC2) is expressed exclusively at satellite cells and represses, through its repressive mark H3K27me3, muscle differentiation genes [119]. ChIP sequencing experiments revealed that EZH2 and SUZ12 bind at the promoters of master regulators of myogenesis, such as MYOG, to repress their expression [120]. During maturation of muscle cells, EZH1 replaces EZH2, although SUZ12 remains bound to the MyoG promoter. The EZH1 binding enhances the RNA polymerase II elongation mechanism and promotes myogenesis in satellite cells [21,120]. Therefore two forms of PRC2 containing either EZH2 or EZH1 might play distinct roles in undifferentiated versus differentiated myocytes, similar to what has been observed in the epidermis. In this sense, deletion of EZH2 would promote the recruitment of EZH1 to differentiation genes, explaining why it results in the rapid differentiation of muscle stem cells. Opposite to the effect of deletion of EZH2, loss of BMI1 or RING1B (PRC1) in muscle stem cells results in an impairment of myogenesis [121,122]. Interestingly, a recent report has shown that with the progressive lost of BMI1 the muscle stem cells from geriatric mice enter to a pre-senescence phase that irreversibly prevents their entry into the cell cycle in a p16Ink4a-dependent manner [123]. The machineries involved in DNA methylation and demethylation are also important for myogenesis. The genes encoding the master regulators of myogenesis such as MyoG and MyoD are highly DNA-methylated in satellite cells. Upon myogenesis, the levels of the maintenance DNA methyltransferase DNMT1 is reduced, and DNA methylation levels are decreased in these differentiation genes [124]. Thus loss of DNA methylation during muscle lineage commitment is


9


caused by passive DNA demethylation, although it can also occur by active DNA demethylation. In this sense, the expression of TET1 and TET2 is upregulated during myogenesis, and the levels of hydroxymethylation levels are globally increased in myoblasts compared with muscle progenitors [124]. Epigenetics mechanisms described in other adult tissues The role of epigenetic factors in the homeostasis of other tissues is starting to be understood. For instance, the histone H3K9 dimethyltransferase G9a is essential for adipose tissue homeostasis. G9a is highly expressed in pre-adipocytes where it represses the expression of adipogenic genes, and its expression is downregulated during adipogenesis. It has been shown that G9a can repress adipogenesis through two independent mechanisms: (a) through the deposition of the H3K9me2 mark on the promoter of the PPAR gene, one of the master regulators of adipogenesis, and (b) by directly promoting the expression of the adipogenesis repressor WNT10a [125]. Accordingly, deletion of G9a in preadipocytes results in an increase of adipogenesis, and deletion in mouse adipose tissue stimulates adipogenic gene expression and increases fat accumulation and tissue weight [125]. Interestingly, the deletion of the histone methyltransferase domain of the thritorax protein MLL3 reduces adipose tissue accumulation in mice [126]. Taken together these two studies suggest that the balance between the activities of MLL3 and G9a regulates adipogenesis and fat accumulation in mammals. Intriguingly, studies aimed at determining changes in DNA methylation at single base resolution have revealed that DNA methylation barely changes as stem cells from the intestinal epithelium differentiate into the different lineages of the villi [37]. As mentioned above, a similar observation was made on comparing quiescent and actively proliferating hair follicle stem cells, suggesting that changes in DNA methylation might not be involved in the function of all adult stem cells [37]. In fact, conditional deletion of DNMT1 results in a modest enlargement of the small intestine crypt zone [127]. However, deletion of DNMT3B in the intestinal epithelium does not result in any phenotype, although DNMT3B expression is required for intestinal tumorigenesis [128,129]. In addition, intestinal stem cells and their differentiated progeny do not show strong differences between histone modifications at enhancers. These sustained active enhancers are thought to allow a continuous cross-talk between adjacent cells promoting differential cell fate [130]. Thus, interestingly these independent studies suggest that 10


DNA methylation and histone modifications are quite static during intestinal stem cell differentiation. That said, there are a few DMRs upon intestinal stem cell differentiation which precisely coincide with enhancers that drive the expression of differentiation genes [37,127]. These cis elements are tissue-specific DMRs, short genome elements that are normally hypomethylated in a tissue-specific manner [46]. Although intestinal deletion of DNA methyltransferases does not result in any overt phenotype, they do profoundly affect intestinal tumorigenesis, suggesting that these DMRs might be important for regulating intestinal stem cell function when tissue homeostasis is perturbed [128,129].

Concluding remarks In the last few years, our understanding of epigenetic function in adult stem cell homeostasis has considerably increased. The advance of next generation sequence technologies is bringing new insights to epigenetic mechanism dynamics. The continuous improvements to next generation sequence techniques are allowing the fields of epigenetics and stem cells to analyse in an unprecedented depth the genetic and epigenetic changes that adult stem cells undergo to sustain homeostasis. Although every tissue-specific stem cell relies on unique epigenetic factors and transcription factors to regulate its function, a common theme is arising from all these studies, one in which the balanced interplay between different epigenetic complexes, or even the same complex formed by combinations of different subunits, is essential for the robustness of the response of adult stem cells to the needs of the tissue at any given time. That said, we are still far from understanding how precisely the marks deposited by these epigenetic factors correlate with the phenotypes observed upon modulation of their expression. Targeted mutations that affect their enzymatic activity in vivo in a tissue-specific manner will be necessary regarding this. In addition, it will be necessary to determine how different chromatin marks established by different complexes are dynamically combined to fine-tune the function of adult stem cells. Notwithstanding this, their importance in tissue homeostasis is not only substantiated by loss and gain of function studies in vivo but is also underscored by the fact that epigenetic regulators are among the most mutated or misregulated genes in human cancers and aged tissues [131]. For instance, changes in the levels of DNA methylation have recently been proposed to be an effective measurement for determining cell ageing [132]. Members of the PRC1 and PRC2 complexes, FEBS Journal (2014) ª 2014 FEBS


such as Ezh2 and Bmi1, or other histone methyltransferases including MLL and G9a, show different kinds of mutations in solid tumours and blood malignancies. In fact, some of these proteins such as MLL or Ezh2 are among the most mutated genes in human cancers and strongly act as oncogenes when overexpressed [133–136]. Interestingly, some of these proteins are susceptible to pharmacological modulation, and inhibitors are already available that might provide new promising antitumoral strategies [137,138].

Acknowledgements This work has been funded by an ERC Starting Grant (STEMCLOCK) granted to SAB. LR is supported by the International PhD La Caixa Fellowship.

Author contributions Lorenzo Rinaldi and Salvador Aznar Benitah discussed the topic of the review, wrote and revised the manuscript.

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Epigenetic regulation of hematopoietic stem cell aging.

Epigenetic regulation in adult stem cells and cancers.

Mathematical model of adult stem cell regeneration with cross-talk between genetic and epigenetic regulation.

Epigenetic control of adult skeletal muscle stem cell functions.

Metabolic regulation of stem cell function.

Role of epigenetic reprogramming in hematopoietic stem cell function.

Epigenetic Regulation of Hematopoietic Stem Cells.

Epigenetic regulation of adult neural stem cells: implications for Alzheimer's disease.

Epigenetic Regulation Shapes the Stem Cells State.

Epigenetic regulation in stem cell development, cell fate conversion, and reprogramming.

Epigenetic regulation of miRNA-cancer stem cells nexus by nutraceuticals.

Epigenetic regulation of open chromatin in pluripotent stem cells.

Epigenetic Regulation of Epidermal Stem Cell Biomarkers and Their Role in Wound Healing.

Epigenetic Mechanisms Regulating Mesenchymal Stem Cell Differentiation.

Histone H1-mediated epigenetic regulation controls germline stem cell self-renewal by modulating H4K16 acetylation.

Dissecting integrin-dependent regulation of neural stem cell proliferation in the adult brain.

Regulation of haematopoietic stem cell proliferation by stimulatory factors produced by murine fetal and adult liver.

FDA regulation of adult stem cell therapies as used in sports medicine.

Dynamics of epigenetic age following hematopoietic stem cell transplantation.

Dynamics of epigenetic regulation at the single-cell level.

Biophysical regulation of epigenetic state and cell reprogramming.

Epigenetic regulation of IL-12-dependent T cell proliferation.

Translational research of adult stem cell therapy.

Epigenetic Regulation of Adipokines.