Content for this white paper was derived from an advisory board meeting of experts in epigenetics and/or skin aging as well as from the literature. This white paper was supported by Erno Laszlo, Inc.

Advisory Board Meeting Participants and Affiliations

Brian Capell MD, PhD (University of Pennsylvania), Raymond Cho MD, PhD (University of California at San Francisco), Manel Esteller MD, PhD (Josep Carreras Leukaemia Research Institute), Elena Eshkova PhD (Icahn School of Medicine at Mount Sinai), Taihao Quan MD, PhD (University of Michigan), Jouni Uitto MD, PhD (Thomas Jefferson University), Christos Zouboulis MD (Dessau Medical Center, Brandenburg Medical School Theodor Fontane)

Introduction

Genetic, environmental, and lifestyle factors interact to result in the accumulation of macromolecular damage and molecular deficits that manifest as aging. Studies of diseases of aging have definitively shown that genetic changes alter the behavior of cells; this process often begins with mutations that inactivate normal cellular mechanisms for monitoring the fidelity of DNA replication, resulting in the rapid accumulation of mutations in genes involved in controlling the growth and death of cells. Similarly, aging has long been known to be associated with the accumulation of mutations, leading to cellular dysfunction and, ultimately, to a senescent phenotype (Lee 2018). On the single-cell level, lifespan has been shown to decrease in a logarithmic fashion as mutation burden increases (Lee 2018).

It has been increasingly recognized that other types of alterations in the genome, known as epigenetic changes, can modulate its structure and function without affecting the underlying DNA sequence. The term “epigenetics” was initially defined by Conrad Waddington in the 1940s, but in the modern context, it was formally defined in the 1990s by Wolffe and Matzkeset as “the study of heritable changes in gene expression that occur without a change in DNA sequence” (Alokail 2015). Epigenetic alterations, acting both independently and together with increasing mutational burden, genomic instability, and stem cell exhaustion, can influence gene expression in ways that promote aging (Saul 2021). Each of the 3 major epigenetic regulatory pathways—DNA methylation, chromatin dynamics (histone modifications), and expression of noncoding RNAs—has been shown to be associated with changes in cellular and organ functions as organisms age (Alokail 2015; Saul 2021). The skin, as the protective envelope of the body, is exposed to multiple environmental insults. Perhaps the most prominent of these is ultraviolet sun radiation. However, other factors such as pollution also have a profound impact on the structure and function of the skin. Recent data show that these extrinsic environmental factors synergize with intrinsic age-related changes to influence epigenetic regulation of gene expression, ultimately contributing to the visible—and invisible—signs of skin aging (Chevalier 2019). This white paper will first provide a brief refresher on the central paradigm of molecular biology, the rigorously controlled process by which genetic information flows within cells and biological systems. Subsequent sections focus on the epigenetic regulation of gene expression and its relevance to skin aging.

1. A REVIEW OF FUNDAMENTAL CONCEPTS IN MOLECULAR BIOLOGY

It is important to have a fundamental understanding of how information flows from gene to protein to understand the role of epigenetics in aging.

The Central Paradigm of Molecular Biology

Setting aside sporadic mutations, every somatic cell in the body contains an identical genome with an identical complement of genes, each of which encodes a specific protein. However, cell types are differentiated by their program of gene expression. When a signal is sent to express a specific gene, the DNA sequence encoding that gene is used as a template to produce single-stranded RNA in a process called transcription. After transcription, the pre-mRNA transcript is spliced, joining coding exons together while excising introns and generating messenger RNA (mRNA), which is exported from the nucleus and read by ribosomes to produce polypeptides that fold into the final 3-dimensional structure of proteins (Figure 1.1). Although the DNA content of all somatic cells is identical, different cells appear and behave differently. Thus, gene expression must be tightly regulated so that only appropriate genes are expressed in a particular cell type. To accomplish this, the transcriptional unit is preceded by regulatory elements, such as promoters and enhancers, that modulate production of its protein encoding transcript (Figure 1.2). All protein-coding genes require a promoter, a regulatory element that is necessary to initiate the process of transcription. The core promoter is a short length of noncoding DNA that overlaps the transcription start site. Many genes have additional regulatory elements such as enhancers, repressors, or insulators (Allison 2017). Finally, epigenetic mechanisms can also play a role in the modification of gene expression and protein synthesis.

Figure 1.1 Flow of genetic information in a cell

Figure 1.2 A typical gene and regulatory regions (Allison 2017)

Packaging of the Eukaryotic Genome

The term “genome” refers to the totality of DNA in the nucleus. All DNA in the cell’s nucleus is organized into chromosomes—thread-like structures of nucleic acids and proteins. The human diploid genome contains approximately 6 billion base pairs. The total length of DNA is about 2 meters, all of which must be accommodated in the nucleus (Annunziato 2008). To accomplish the necessary compaction, negatively charged DNA is complexed with positively charged histones to form nucleosomes, each of which is composed of 8 histone proteins (2 copies each of H2A, H2B, H3, and H4) wrapped about 1.65 times by DNA. (Figure 1.3). Additional compaction is achieved by a series of folding and coiling steps, ultimately resulting in the chromatid of a chromosome. The compression and unwinding of DNA are tightly regulated through processes that can “tighten” or “loosen” chromatin, allowing regions of the genome to be accessed for transcription.

2. AN INTRODUCTION TO EPIGENETIC REGULATION OF GENE EXPRESSION

Consider that all cells have the same DNA but organisms contain many different types of cells: skin cells, neurons, liver cells, pancreatic cells, inflammatory cells, and many others. The ability of many different cell types to be derived from the same instruction set—the cell DNA— is driven by differential gene expression. Therefore, different subsets of genes are expressed depending on cell type. Epigenetic mechanisms are one way in which gene expression programs are managed in a temporally and spatially appropriate manner to determine cell type, govern cellular behavior, and drive cells along specific differentiation pathways (Simmons 2008). Broadly, there are 3 epigenetic pathways that regulate gene expression: DNA methylation, histone modifications, and RNA-associated silencing (Simmons 2008).

• DNA methylation involves the addition of a methyl group to DNA. It primarily occurs in region tracts of DNA called CpG islands, where cytosine nucleotides alternate with guanine nucleotides.

Figure 1.3 DNA is packaged in multiple steps to compress its 2-meter length into a structure that can fit into the cell nucleus (Annunziato 2008)

• Histone modifications involve posttranslational modifications that alter chromatin structure. Some of these “loosen” chromatin, allowing it to be accessed by transcriptional machinery. Other modifications condense chromatin into heterochromatin, which is not transcriptionally active. Acetylation and methylation are the primary, but not the only, epigenetic mechanisms governing chromatin dynamics.
• Genes can be silenced by antisense RNA. RNA may also affect gene expression by causing heterochromatin to form, triggering histone modifications, or by modulating DNA methylation.

DNA Methylation

Methylation of the DNA base cytosine is the major DNA modification in most animals and plants (Allison 2017).

Cytosine DNA methylation is a covalent modification of DNA. In this reaction, a methyl group (CH3) is transferred from s-adenosylmethionine (SAM) to the carbon-5 position of cytosine by cytosine DNA methyltransferases (Dnmt). 5-methyl-cytosine is the only modified base in most cells (Allison 2017).

DNA methylation occurs almost exclusively at the dinucleotide CG in mammals, often denoted as “CpG,” where p stands for the phosphate group. CpGs can occur in islands consisting of multiple CpG repeats. These islands are found in the promoter regions of about half of the genes in the human genome, where they are sparsely methylated. In contrast, 80% of CpGs outside these islands are heavily methylated (Alokail 2015).

Importantly, methylation is maintained during DNA replication, so daughter cells retain the methylation patterns of the parent cell. After replication, the DNA double helix is hemimethylated—the template strand retains its original methylation pattern, and the newly synthesized strand is unmethylated. A maintenance DNA methyltransferase, DNMT1, recognizes only hemimethylated sites and methylates the new strand of DNA appropriately (Allison 2017).

Methylation is usually, but not always, a way of marking genes for silencing, although the precise effect varies by the location of the methylated DNA region (Figure 2.1): (Aquino 2018)

• Methylation in the promoter region shuts off gene expression
• Methylation of the transcription initiation site also shuts off gene expression
• Methylation inside the protein-coding region gene is often correlated with increased gene expression

Histone Modification

Histone modifications are important in transcriptional regulation. DNA that is wrapped tightly around histones is not accessible to the transcription machinery. To make it accessible, the region wrapped around histones must “relax.” The compressed state is referred to as “heterochromatin” and the relaxed state as “euchromatin.” The transitions between heterochromatin and euchromatin are accomplished by enzymatic modification of histones. This process is tightly regulated to control gene expression and is highly tissue-specific, with each tissue displaying a characteristic pattern of transcriptionally accessible regions (Simmons 2008). The N-terminal tails of histones, which protrude from the core histone octamer, are subject to at least 6 different types of covalent modifications: acetylation, methylation, ubiquintinylation, phosphorylation, ADPribosylation, and sumoylation. These modifications control access to the DNA wrapped around the histone (Allison 2017). The enzyme histone acetyltransferase (HAT) adds up to 3 acetyl groups to ≥1 of the lysine residues in histone tails (Alokail 2015). The addition of a negatively charged acetyl group reduces the overall positive charge of histones, reducing the affinity of histone tails for negatively charged DNA. The action of HAT is countered by histone deacetylase (HDAC), which removes acetyl groups from lysine in histone tails. Acetylation of histones is generally associated with active regions of gene expression, while deacetylation generally correlates with regions of reduced gene expression (Simmons 2008). Methylation and demethylation are catalyzed by histone methyltransferases (HMTs) and histone demethylases (HDMs), respectively (Figure 2.2). Methylation of histones has a more complex set of outcomes than acetylation; while methylation of certain residues can suppress transcription, methylation of other residues may have an activating effect.

Figure 2.1 The effects of DNA methylation vary by location (Aquino 2018)

Figure 2.2 Methylation of histones can “relax” chromatin, allowing the transcriptional machinery to access DNA regions

RNA-Associated Silencing

As discussed earlier, mRNA carries genetic information from DNA to the ribosome, where it is translated to proteins and is thus referred to as coding RNA (RNA that codes for proteins). Large regions of the genome do not code for proteins and were long regarded as “junk” DNA. It was not until the 1990s that a role for this DNA was elucidated. Today, it is known that many types of RNA are transcribed from these DNA regions but are never translated into proteins. These RNAs—referred to collectively as noncoding RNAs—function as posttranscriptional epigenetic regulators of gene expression (Chhabra 2017). There are at least 5 classes of noncoding RNAs that play roles in mammalian cells, including but not limited to regulation of apoptosis, signaling, embryonic development, and tissue differentiation (Table 2.1).

Type	Length	Functions	Number in Humans
MicroRNA (miRNA)	19-24	mRNA degradation or repression of translation	-2500
Short-interfering RNA (siRNA)	20-25	mRNA degradation	Unknown
Piwi interacting RNA (piRNA)	26-31	Transposon silencing, germline development	-23,000
Small nucleolar RNA (snoRNA)	60-150	Modification of rRNA	-400
Long noncoding RNA (lncRNA)	>200	Chromatin reprogramming, precursors of small RNAs	-120,000

Table 2.1 Classes of noncoding RNAs associated with epigentic phenomena (Chhabra 2017)

 

Figure 2.3 miRNA transcription, processing, and mechanism of action (Magri 2017)

miRNAs are transcribed from DNA by RNA polymerase 2 and then undergo a number of processing steps before being loaded into the RNA-induced silencing complex, or RISC, which utilizes the noncoding RNA to recognize target sequences on mRNA. If the RISC complex encounters a perfectly complementary sequence, the target mRNA is cleaved. If the RISC complex has partial complementarity to the target, translation of the target mRNA is suppressed (Magri 2017, p 3A) (Figure 2.3).

3.THE EPIGENETICS OF SKIN AGING

Skin Aging

The skin is the largest organ of the body. Because it is continuously exposed to environmental insults, efficient and continuous replacement of its outer layers is critical for proper function.

The epidermis is a relatively thin layer composed of epithelial cells that serves as a protective shield, while the underlying dermis—which makes up the bulk of the skin— is composed of dense, connective tissue penetrated by nerves and blood vessels from its lower side and skin appendages (pilosebaceous units and sweet glands) from its upper side. The epidermis itself has a layered structure characterized by progressive differentiation of cells as they move toward the surface. The stratum basale, the deepest layer of the epidermis, is composed of a single layer of undifferentiated epithelial cells, with a few progenitor cells among them, that continuously divide to produce differentiating keratinocytes. As these cells divide, they push cells upward to begin differentiation into keratinocytes and, ultimately, into the heavily keratinized anucleate corneocytes that form the stratum corneum. The dermis contains fibroblasts that produce an extracellular matrix composed of collagen, elastic fibers, glycoproteins, and proteoglycans, which together maintain skin architecture and confer elasticity, resistance, and strength to the tissue (Orioli 2018; Marieb 2015; Kang 2018).

The cells of the epidermis and dermis are characterized by tightly controlled programs of gene expression. During epidermal self-renewal, each stem cell must choose between divisions that result in self-renewal or divisions that result in a daughter cell that is fated to undergo terminal differentiation (Zouboulis 2008). Once this choice has been made, a complex series of temporal and spatial changes in gene expression occur as the keratinocyte progressively differentiates and ultimately ejects its nucleus to become a heavily keratinized corneocyte (Orioli 2018). In the dermis, tightly regulated gene expression programs manage the maintenance of epidermal homeostasis, wound healing, and production of the extracellular matrix, among other activities. (Orioli 2018; Kang 2018)

Both intrinsic and extrinsic factors synergize to produce skin aging (Zouboulis 2011; Figure 3.1). Young, healthy skin is characterized by a thick, well-organized dermal layer abundantly populated by fibroblasts producing elastic fibers and collagen. In aged skin, dermal fibroblasts become senescent, resulting in reduced production of elastic fibers and a loose, disorganized, and degraded collagen structure.

Figure 3.1 Morphologic features of young, aged, and photoaged skin (Orioli 2018)

At the same time, the epidermis thins, resulting in the appearance of wrinkles. As with intrinsic aging, extrinsic aging of the skin—such as that caused by photodamage or pollutants—is characterized by dermal atrophy. In contrast, photoaging is associated with a heterogeneously thickened epidermal layer that contributes to skin wrinkling (Orioli 2018).

Role of Epigenetics in Skin Aging

Modifications in all 3 epigenetic pathways—DNA methylation, chromatin dynamics, and RNA-based silencing—are seen in aging skin. Due to the sheer abundance of data on this topic, this section will only touch on some examples of changes in aging skin. Suggestions for further reading are provided at the end of this white paper for those interested in more detail.

DNA Methylation

DNA methylation patterns in epidermal and dermal tissue have been shown to change with aging. While methylation patterns are similar in young individuals, they display increasing divergence as organisms age, suggesting a role for environmental factors in modulating methylation marks (Orioli 2018). Increased methylation heterogeneity has been linked to cellular senescence, at least in vitro, and has been demonstrated in aging fibroblasts (Koch 2011). Expression of the DNA methyltransferase DNMT1 decreases as fibroblasts are sequentially passaged, and experimental silencing of DNMT1 in young fibroblasts induces a senescent phenotype (De Paoli-Iseppi 2017; Lopatina 2002; Wang 2017).

Methylation patterns change over time in multiple genes implicated in dermal aging. For example, the TET2 gene is hypermethylated in epidermis samples from elderly individuals (Gronniger 2010). Recent data suggest that TET2, which is itself an epigenetic regulator, has roles in modulating cell viability, apoptosis, and the expression of inflammatory mediators in keratinocytes (Liu 2020). Similarly, DDAH2, an enzyme in the nitric oxide pathway, is hypermethylated in aging and chronically sun-exposed tissues (Gronniger 2010). Downregulation of DDAH2 may predispose the keratinocytes cell to accelerated oxidative damage.

The evidence for an impact of extrinsic factors, such as ultraviolet radiation, on DNA methylation is inconsistent. Some studies have shown distinctive patterns of hyperand hypomethylation in photoaged tissues, while others have not demonstrated a consistent link (de Olivera 2020).

Chromatin Dynamics

Aging is associated with changes in chromatin organization. In particular, aging is associated with the replacement of canonical histones by histone variants. For example, the histone variant H2A.J—which differs by only a single amino acid from the canonical H2A histone—is expressed and incorporated into the chromatin of human fibroblasts during senescence, which allows the transcriptional machinery to constitutively access previously tightly regulated inflammatory genes (Contrepois 2017). Further, H2A.J is deposited in response to DNA damage induced by ionizing radiation (Isermann 2020).

H3K27me3, denoting the addition of 3 methyl groups to lysine (K) 27 on histone H3, is currently the beststudied histone modification with a potential role in skin aging (Orioli 2018). This mark, which is associated with transcriptional suppression of nearby genes, is added by the methyltransferase Polycomb Repressor Complex (PRC) and removed by Jumonji family demethylases (Leon 2019). PRC manages epidermal stem cell identity and self-renewal by suppressing nonlineage, differentiation, and senescence genes; reduced expression of subunits of this complex have been linked to skin aging and senescence of both keratinocytes and fibroblasts (Perdigoto 2014; Sen 2008). PRC activity is directly affected by UVA radiation, resulting in reduced hyaluronic acid production by and senescence of human dermal fibroblasts (Orioli 2018). The Jumonji demethylases have also been strongly implicated in fibroblast senescence (Orioli 2018).

Changes in histone acetylation over time are also strongly implicated in skin cell aging. The sirtuins (SIRTs 1–7) are nicotine adenine dinucleotide (NAD)-dependent enzymes involved in managing energy metabolism and oxidative stress, response to UV damage, and inflammation, among other activities. SIRTs 1–6 are involved in the deacetylation of histones, and they also regulate the acetylation status of transcription factors. SIRT1, in particular, has been shown to play critical roles in the regulation of skin homeostasis, including the induction of keratinocyte proliferation, inhibition of epidermal cell senescence, and stimulation of type 1 collagen in fibroblasts. SIRT1 expression is downregulated in aging epidermal tissues. SIRT6, which is thought to manage access of DNA repair proteins to chromatin, is also downregulated in aging keratinocytes and fibroblasts, resulting in increased susceptibility to DNA damage (Orioli 2018).

RNA-Mediated Silencing

While there are a number of different noncoding RNA species that play a role in gene regulation, at present, miRNAs are the best studied in aging.

Differential expression of miRNAs has been demonstrated in aging tissues. In one study that evaluated miRNA expression in the whole blood of over 5000 adults, 127 miRNAs were identified that were differentially expressed based on age (Huan 2018). Of particular importance, the microRNA miR-217, which targets DNMT1 RNA, has been shown in vitro to promote senescence in human fibroblasts by suppressing DNMT1 expression (Wang 2017). In vivo, skin samples derived from patients of different ages showed that miR-217 expression was significantly upregulated in skin tissues from older ndividuals (Wang 2017). miRNAs play direct roles in the post-transcriptional regulation of extracellular matrix proteins, including but not limited to collagen type I and type IV, decorin, fibronectin, and epidermal growth factor (Maurer 2010; Kwan 2015; Shan 2009; Giles 2011).

Many other miRNA-mediated regulatory pathways are important in aging-related gene networks. Members of the miR-30 family are overexpressed in skin aging. This family of microRNAs is associated with autophagy, a catabolic process involved in the degradation of worn, abnormal, or malfunctioning cellular components in lysosomes. Autophagy declines in effectiveness as tissues age and may allow for the accumulation of nonfunctional cellular debris that contributes to the aging process. Similarly, members of the miR-200 family are overexpressed in aging tissues. These miRNAs help regulate the oxidative balance and overexpression allows for the accumulation of reactive oxygen species, disturbing skin homeostasis, promoting DNA damage, and accelerating apoptosis. Finally, expression of the miR-181 family is induced in aging skin, where they act as repressors of SIRT1 and also disrupt mitochondrial function (Chevalier 2019). Other miRNAs may influence sebaceous lipogenesis, which generally declines with age (Schneider 2013).

miRNA expression may be a critical link between UV exposure and skin aging. Exposure of cultured keratinocytes to UVB radiation results in >2-fold changes in the expression of 44 miRNAs (Zhou 2012). Further, miRNA expression is involved in the regulation of several aspects of the DNA damage response to promote cell survival after UV exposure. Experimental knockdown of components of the miRNA processing pathway was shown to compromise the checkpoint response and, ultimately, the survival of UV-exposed cells (Syed 2014). At least 5 miRNAs are upregulated in photo-aged skin, including miR-101, which silences translation of EZH2, a subunit of PRC (Greussing 2013; Syed 2014).

4. CONSIDERATIONS FOR PRODUCT DEVELOPMENT AND THE PHORMULA 3-10 COLLECTION

As products are developed to address the appearance of skin, it is important to understand the science behind the signs of aging skin, including the contribution of epigenetics. At Erno Laszlo, we consider this science in the development of our products and in particular our new Phormula 3-10 line.

These Phormula 3-10 products all contain Erno Laszlo’s proprietary complex, Epigene-6, and are formulated to minimize the appearance of aging by providing an optimal environment for the skin to repair itself. Epigene-6 encapsulates three key active ingredients, apple, ginger and niacinamide, to support skin exposed to environmental triggers and target the six signs of aging skin: wrinkles, elasticity, firmness, skin tone, skin hydration, and skin texture.

The Phormula 3-10 line includes Phormula 3-10 Repair and Phormula 3-10 Eye Intensive products:

• Phormula 3-10 Repair is an advanced nurturing balm with Epigene-6, niacinamide and the skin conditioning Cera-Skin complex to provide a nourishing environment for the skin to accelerate the recovery of a compromised skin moisture barrier. It has been clinically tested and shown to significantly decrease the appearance of wrinkles, and improve elasticity, firmness, skin tone, skin moisture barrier, hydration and texture.
• Phormula 3-10 Eye Intensive is a multiaction eye cream with Epigene-6 as well as hawthorn and jasmine flower extracts to improve the appearance of fine lines and wrinkles. Phormula 3-10 Eye Intensive has also been clinically tested and shown to significantly decrease the appearance of wrinkles and eye bags while improving skin hydration around the eye area.

 5. CONCLUSIONS

Epigenetic mechanisms have long been understood to play a critical role in age-related diseases such as cancer, and treatments directed at modulating the activity of various elements of epigenetic pathways are becoming increasingly common in oncology. The skin is not only subject to intrinsic aging but is also exposed to numerous extrinsic insults. Data suggest that the confluence of these factors results in changes in the epigenetically controlled expression of genes that play roles in skin homeostasis. The disruption of these pathways likely contributes to the visible manifestations of aging. As products are developed, it is important to consider the science of aging.

6. FURTHER READING

The role of epigenetics in skin aging is complex. For those who are interested and would like to read further, we recommend the following reviews.

Chevalier FP, Croteau J, Lamartine J. MicroRNAs in the functional defects of skin aging. In: Noncoding RNAs. InTech Open. 2019.

Orioli D, Dellambra E. Epigenetic regulation of skin cells in natural aging and premature aging diseases. Cells. 2018;7:1-30.

Syed DN, Khan MI, Shabbir M, et al. MicroRNAs in skin response to UV radiation. Curr. Drug Targets. 2013;14:1128- 1134.

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