Epigenetic and metabolic regulation of epidermal homeostasis

Abstract Continuous exposure of the skin to environmental, mechanical and chemical stress necessitates constant self‐renewal of the epidermis to maintain its barrier function. This self‐renewal ability is attributed to epidermal stem cells (EPSCs), which are long‐lived, multipotent cells located in the basal layer of the epidermis. Epidermal homeostasis – coordinated proliferation and differentiation of EPSCs – relies on fine‐tuned adaptations in gene expression which in turn are tightly associated with specific epigenetic signatures and metabolic requirements. In this review, we will briefly summarize basic concepts of EPSC biology and epigenetic regulation with relevance to epidermal homeostasis. We will highlight the intricate interplay between mitochondrial energy metabolism and epigenetic events – including miRNA‐mediated mechanisms – and discuss how the loss of epigenetic regulation and epidermal homeostasis manifests in skin disease. Discussion of inherited epidermolysis bullosa (EB) and disorders of cornification will focus on evidence for epigenetic deregulation and failure in epidermal homeostasis, including stem cell exhaustion and signs of premature ageing. We reason that the epigenetic and metabolic component of epidermal homeostasis is significant and warrants close attention. Charting epigenetic and metabolic complexities also represents an important step in the development of future systemic interventions aimed at restoring epidermal homeostasis and ameliorating disease burden in severe skin conditions.

stem cells (EPSCs) reside in the basal layer of the epidermis where they are attached to the basal membrane, which separates the epidermis from the underlying dermis. As stem cells differentiate, they move upward through the different layers of the epidermis towards the surface of the skin. 1 The rate by which adult epidermal stem cells renew themselves and yield daughter cells depends on developmental stage, external injury, steady-state tissue turnover and remodelling. Several models of epidermal differentiation and regeneration have been posited to explain the nature and behaviour of EPSCs located within the basal layer of the epidermis. 3,4 The hierarchical model of epidermal homeostasis proposes the existence of a limited number of slowcycling long-term stem cells within the basal layer that self-renew and give rise to fast-cycling transit-amplifying cells. 5 According to the stochastic model, on the other hand, all basal cells have equal potential to either divide or directly differentiate. 3,6 The existence of slow-and fast-cycling stem cells that occupy spatially distinct skin regions and are capable of producing unique differentiated lineages suggests yet another possibility. 7 Recent data in human 3D cultures suggest that there is a striking variety of signalling processes in the basal layers of the epidermis despite the relatively stable architecture of the terminally differentiated layers. 8 Which of the different models of stem cell differentiation and regeneration most accurately describes EPSC behaviour in vivo is still a subject of ongoing research. 9 Through control of gene expression and homeostasis, aspects of the epigenome regulate almost every biological process, from cellular differentiation and maintenance of phenotypes to onset of disease and ageing. 13,14 Epigenetic mechanisms such as DNA methylation, histone tail modifications, chromatin accessibility and changes in DNA architecture are tightly correlated with normal cellular function, while their dysregulation manifests in aberrant gene expression and disease. 15 According to a contemporary definition, epigenomics is defined as "the study of molecules and mechanisms that can perpetuate alternative gene activity states in the context of the same DNA sequence". 14 Because of their essential role in establishing specific transcriptional configurations, epigenetic mechanisms govern many aspects of EPSC proliferation, as well as differentiation of their descendants. [16][17][18][19] Uncharacteristic epigenetic modifications often associate with a loss of transcriptional fidelity, unchecked proliferation, de-differentiation, and malignant epidermal to mesenchymal transition. 20 At the same time, pronounced changes in the epigenetic landscape often accompany, and are critical for, resolving challenges to epidermal homeostasis induced by changes in the local microenvironment or external stimuli, such as injury. 21 Ageing and a variety of diseases, such as chronic inflammation or cancer, manifest themselves in characteristic changes of the epigenetic profile. [22][23][24] A multitude of other factors, ranging from DNA damage to dietary-or drug-induced metabolic changes, are known to affect the epigenetic status as well. For example, exposure to high altitude, 25,26 cancer-associated elevated concentration of lactate, 27 and increased uptake of dietary methionine 28,29 have been linked to epigenetic changes and thus highlight the intricate connection between metabolic events and alterations in the epigenome. The pivotal role of mitochondrial energy metabolism in regulating epigenetic events and epidermal homeostasis will be discussed below.
The epigenome is structured into distinct, but interconnected layers ranging from overall chromatin structure and organization to specific histone and DNA modifications. Histones are predominantly modified by methylation, acetylation and phosphorylation, but they can be adapted by other modifications such as ubiquitination, sumoylation, ribosylation and citrullination. 30 While DNA methylation is the most prominent and studied epigenetic modification (see Box 1|Mechanisms of DNA methylation), other aspects of the epigenome include RNA methylation, 31 41 or the 4D nucleome project. 42 The EWAS data hub -comprising normalized DNA methylation array data from 75 K samples -is now available for epigenome-wide association studies (EWAS). 43

| Epigenetic regulation of epidermal homeostasis
There is ample evidence that epigenetic mechanisms, such as DNA methylation, histone modifications or changes in DNA topology, contribute to epidermal homeostasis and differentiation [49][50][51][52] (summarized in Figure 1 and Table 1). Epigenetic regulation has also been analysed in wound healing and functional links between chromatin architecture and gene expression in keratinocytes have been found. [53][54][55][56] Disrupted chromatin regulation, prompted by the loss of PRC1, results in impaired epidermal tissue integrity and blistering skin resembling human skin fragility syndromes. 49 In regards to histone modifications, chemical inhibition of histone demethylases impairs differentiation of inter-follicular stem cells and delays injury repair. 50 Chronic sun exposure is associated with distinct histone acetylation changes and altered gene expression in human photodamaged skin. 57 Histone acetyltransferase (HAT) activity is dependent on zinc and depletion of zinc results in decreased HAT activity. The epithelial zinc transporter ZIP10 epigenetically regulates human epidermal homeostasis by modulating zinc availability and histone acetyltransferase activity. 58 Reduced ZIP10 activity or depletion of zinc leads to reduced HAT activity and decreased expression of genes, such as filaggrin or metallothionein, involved in epidermal homeostasis. 58 Likewise, dynamic epigenetic regulation of DNA methylation (see Box 1|Mechanisms of DNA methylation) is critical for the maintenance of EPSC status and proliferative capacity. A progressive loss of DNA methylation patterns caused by forced depletion of DNMT1 in the epidermis leads to failure of EPSC self-renewal and tissue regeneration. 46 Consistently, DNMT1 expression is normally restricted to the basal layers of the epidermis containing the EPSC population, and mostly absent in the outer differentiated layers. The de novo DNMTs, DNMT3A and DNMT3B, also critically contribute to EPSC homeostasis by controlling enhancer methylation and active chromatin conformation of stem cell relevant genes. 59 Specifically, co-localization of DNMT3A and TET-2 at target enhancers results in 5-hmC formation and gene activation. 59 Interestingly, DNMT3A and DNMT3B also seem to protect the epidermis from tumourigenesis since the loss of these genes in the mouse epidermis promotes squamous transformation. 60 In atopic dermatitis, DNA methylation patterns from patients differ significantly from those of healthy controls. 61

| The role of miRNAs in epidermal homeostasis
The overall contribution of miRNAs to skin homeostasis was demon- MiRNome profiling, coupled with functional validation of candidates, continues to drive our understanding of miRNA regulation of prominent skin processes, both in health and disease contexts (see Table 2). In the context of epidermolysis bullosa (EB) -a rare genetic disorder of the skin discussed in more detail below -the role of miR-NAs in disease pathogenesis is beginning to surface. To date, three miRNAs have been described to modulate EB-associated complications such as fibrosis (miR-29b, 86,87 miR-145 88,89 ) and cancer (miR-10b 90 ). The repetitive destabilization of the extracellular matrix that accompanies recessive dystrophic EB (RDEB) upon injury results in progressive soft tissue fibrosis with debilitating consequences, such as tumour development. 91 miR-145-5p was shown to be upregulated in RDEB-fibroblasts, which typically exhibit more contractile features than their wild type counterparts, indicating a potential correlation between RDEB severity and miR-145-5p levels, by contributing to skin fibrosis. 88 Indeed, inhibition of miR-145-5p resulted in a downregulation of α-SMA, TAGLN and JAG1, all of which are contractile markers, leading to a reduction of fibrotic traits. 88 Another miRNA, miR-29, which directly targets the disease-causing gene COL7A1, as well as the essential COL7A1 expression regulator SP1, was found to be downregulated in RDEB fibroblasts. 87 Furthermore, in a complex network, TGF-ß was shown to be a further activator of COL7A1 expression and at the same time reduces miR-29 levels via SMAD phosphorylation. 87,92,93 Apart from COL7A1 regulation, miR-29 family members were also shown to influence DNA methylation by targeting distinct DNA methyl transferases 94 and proteins involved in DNA demethylation. 95 Patients suffering from RDEB are particularly prone to developing exceptionally aggressive squamous cell carcinomas (SCCs). In this context, overexpression of miR-10b has been attributed a role in conferring stemness to tumour cells, specifically by increasing cell adhesion in 2D and 3D functional models. While miR-10b is the first miRNA described to be associated with RDEB-SCCs, 90 the role of miRNAs in tumourigenesis is generally well-accepted, and has been described for several tumour entities, among them cutaneous SCCs, affecting diverse mechanisms like migration and proliferation. 96-98

| Mitochondrial control of epidermal homeostasis
Emerging evidence suggests that mitochondria are vital regulators of skin physiology. 99 Epidermal progenitor/stem cells do not rely F I G U R E 1 Epigenetic effectors and epidermal homeostasis. Maintenance and differentiation of EPSCs critically governs epidermal homeostasis. Individual, proliferating EPSCs (indicated as shaded cells) are located in the basal layer. As cells differentiate, they progressively move upward through the various layers of the epidermis. Eventually, these cells lose their nuclei before forming the layers of the outermost stratum corneum. Multiple epigenetic effectors regulate EPSC selfrenewal, proliferation and differentiation. These factors control DNA methylation (indicated in blue), histone modification and chromatin remodelling (indicated in orange). Abbreviations: BL, basal layer; BM, basement membrane; DNMT, DNA methyltransferase; EPSC, epidermal stem cell; HDAC, histone deacetylases; PRC, polycomb repressive complex; SC, stratum corneum; SG, stratum granulosum; SL, stratum lucidum; SS, stratum spinosum; TET, ten-eleven translocation; TrxG, trithorax group proteins on the mitochondrial respiratory chain, but still require a functional dynamic mitochondrial compartment. 100 One main task of keratinocytes is corneocyte renewal and production of stratum corneumspecific proteins and lipids needed for a functional skin barrier.
These processes require high amounts of energy, which is normally     63 In this publication, differences in DNAmethylation are described for genes that are involved in regulating epidermal homeostasis and innate immunity, i.e. KRT6A

BOX 3 Epigenetic regulation in disorders of cornification
OAS2, S100A and LRRC8C, the latter with expression probes in trans with CD36. In turn, CD36 was shown to be increased in states of skin barrier disruptions 71 and mutations in CD36 cause ichthyosis prematurity syndrome 72 with skin barrier abnormalities and disturbances in epidermal lipid metabolism. 73 In contrast, the level of demethylation of FOX3i1 in circulating

regulatory T cells (Tregs) is similar between AD and control
subjects. 74 However, the demethylation of the FCER1G promoter in monocytes 75 and the TSLP promoter in keratinocytes showed differences. 76 Thus, current knowledge implies epi-

| Epidermal homeostasis in epidermolysis bullosa
Recently, our understanding of EPSC biology and epidermal homeo-   Col17a1 acts as a strong disease modifier. 127 Through the application of combined ex vivo cell and gene therapy, almost the entire epidermis of an EB patient can be reconstituted by genetically corrected long-lived EPSCs. 12,128,129 In a series of therapeutic skin transplantations, we discovered that, apart from technical issues, the outcome of the procedure depends on the anatomical site of the initial biopsy, the age of the patient, the genes involved, and, perhaps more importantly, on the microenvironment characterizing the receiving wound bed.
The contribution of age is common knowledge in the field and has also been observed by us 128 in the case of a 49-year-old patient vs.
a 7-year-old patient. 12 The intrinsic ageing processes of the skin have been revealed to depend on cytoskeletal proteins (e.g. keratins; cytoskeletal proteins including desmosomes, microtubules and microfilaments) 130 and other cellular processes, like cell cycle control, inflammatory response, signalling and metabolism. [131][132][133] Moreover, EB per se is a disease not only of skin attachment, but it also displays an ageing phenotype exemplified by a specific gene expression signature. 134 In the case of JEB, this has been related to dysregulation of the YAP/TAZ pathway, which causes progressive, age-related depletion of stem cells. 135 We provided evidence that the reduction of clonogenic potential and the loss of stem cells in primary JEB keratinocytes is associated with perturbation of the YAP/TAZ signalling which renders ex vivo gene therapy cumbersome. 135 The Hippo signalling pathway, better known for its function in organ size control through its effectors Yes-associated protein (YAP) and WW domaincontaining transcription regulator 1 (commonly listed as TAZ), has been demonstrated to play a pivotal role in regulating tissue homeostasis and regeneration in skin. [135][136][137][138][139] The transcriptional regulators YAP and TAZ localize to the nucleus in the basal layer of human and mouse epidermis 135,139,140 and are elevated during wound healing. [136][137][138][139] Skin specific deletion of both YAP and TAZ in adult mice leads to hair loss and impairs regeneration after wounding. 136 YAP expression correlates with stem cell content and it has been reported that nuclear YAP progressively declines with age and correlates with the proliferative potential of epidermal progenitors. 135,139 Compared to those derived from healthy donors, EPSCs from EB patients are often difficult to culture ex vivo. Repeated wounding and sustained proliferative stress may contribute to decreased plasticity and increased exhaustion of EPSCs in EB patients. There are distinct differences in clonogenic ability and proliferation potential in LAMB3-and COL7-deficient keratinocytes. In LAMB3-deficient keratinocytes, both properties are severely altered, but they can be rescued by transduction with a LAMB3-expressing vector ( Figure 2).  [142][143][144] and resistance to cisplatin treatment. 145 A different study, however, found promoter hypomethylation and up-regulated expression of LAMB3 in gastric cancer. 146 To our knowledge, however, there are currently no published reports on prospective epigenetic differences of EPSCs from EB patients and age-matched healthy donors.
In light of high somatic mutation rates, stem cell competition further appears to be an important factor in maintaining tissue ho-

ACK N OWLED G EM ENTS
We thank Dr. Rudolf Hametner for his help in preparing Figure 1.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.