When Friar Gregor Mendel published his memoir on plant hybridization in 1866, little did he know that his work would lay the foundation for the study of hereditability [1,2]. Unbeknownst to him, Mendel planted the initial seed for what would eventually become one of the primary branches of modern biology: genetics. Today, we all recall our biology lessons from school, where we studied hereditability diagrams illustrating pea coat color and texture to grasp the principles of Mendelian laws and how certain human traits were inherited.

By the first half of the 20th century, geneticists had a thorough knowledge of chromosome morphology, as well as the processes of mitosis and meiosis. They theorized that genes resided on chromosomes, aligning perfectly with Mendelian and hereditability laws. It was not until the 1940s that scientists identified nucleic acids as the carriers of genetic information, although they remained uncertain about how these molecules stored, replicated, and transferred this information to daughter cells following cell division [3]. The emergence of molecular genetics, along with the groundbreaking research of James Watson, Francis Crick, and Rosalind Franklin, unveiled the structure of DNA in 1953, establishing the foundation for comprehending DNA replication and the transfer of genetic information [4,5]. Nearly fifty years later, the Human Genome Project published the complete sequence of the human genome [6].

Nevertheless, the DNA structure, the genetic code, and the mechanisms behind translating genetic information into proteins falls short of explaining many crucial biological processes. For instance, how do embryonic stem cells differentiate and give rise to all the cell types found in an organism during embryogenesis, despite sharing the same genome? How do these cells “know” which cell type they should become? There must be something instructing stem cells on which genes to activate or deactivate to become a differentiated hepatocyte and not an erythrocyte, for example. In simpler terms, there must be a mechanism regulating gene expression. This article delves into precisely that topic: Epigenetics.

In this article, we will explore the concept of epigenetics, learn about its historical development, and examine the primary epigenetic mechanisms operating within a cell.

What is Epigenetics?

The term “epigenetics” is formed by adding the Greek prefix “epi-” (meaning “over,” “outside,” or “around”) to genetics. Defining epigenetics is a complex task, and its definition has evolved over the years. It was first coined in 1942 by the developmental biologist Conrad H. Waddington to refer to a new field of biology concerned with the connections between gene and protein expression [7].

As a developmental biologist, Waddington sought to unravel the mystery of how embryo cells possessing the same genes can develop into diverse tissues in the body. How could something as simple as the genetic code give rise to such a sophisticated organism with an array of distinct cell types? Waddington considered cell differentiation an epigenetic phenomenon, and that cell fate relies on what he called “the epigenetic landscape.” The concept was graphically represented by a ball atop a slope with various potential paths depicted as grooves. The ball could follow any permitted trajectory through the epigenetic landscape, ultimately determining the final outcome or cell fate [8-10].

In the ensuing decades, the notion of epigenetics remained blurry as it evolved and encompassed numerous biological processes. It began to crystallize in the 1970s and 1980s when Robin Holliday and Art Riggs proposed that DNA methylation acts as a form of cell memory [11,12]. They sought to explain why cells remain differentiated through multiple rounds of cell division rather than reverting to undifferentiated states or transforming into another cell type. Since DNA methylation represses gene expression, they proposed that methylation patterns could serve as a mechanism by which cells “remember” their identity and maintain differentiation. Holliday and Riggs introduced the idea of memory into the term epigenetics.

Due to the varied definitions associated with epigenetics, arriving at a concise and universally accepted definition is challenging. For the purposes of this article, we will narrow it down to the following: any change in gene expression that can be heritable, yet is reversible and does not alter DNA sequence [10].

How does Epigenetics work?

As per the preceding definition, epigenetic mechanisms affect gene expression without altering DNA sequence. Additionally, these mechanisms can induce changes that are both reversible and heritable. Let’s delve into the primary epigenetic mechanisms responsible for regulating gene expression!

DNA methylation

The discovery of DNA methylation occurred concurrently with the identification of DNA as the genetic material in the 1940s. Rollin Hotchkiss stumbled upon modified cytosine while studying a preparation of calf thymus using paper chromatography, and he hypothesized that this fraction was 5-methylcytosine (5mC) [13]. It took another forty years for researchers to identify DNA methylation as a mechanism for regulating gene expression and cell differentiation [14].

Enzymes involved in DNA methylation fall into three broad categories based on their functions: writers, erasers, and readers. Writers are responsible for transferring methyl groups to cytosine. Conversely, erasers modify and remove these methyl groups. Readers, on the other hand, identify and bind to methylated regions of the genome, ultimately regulating gene expression.

Methylating and demethylating: Writers and Erasers

DNA methylation is catalyzed by a group of enzymes known as DNA methyltransferases (Dnmts). These enzymes transfer a methyl group from S-adenyl-methionine to the fifth carbon of cytosine, forming 5mC. Dmnt3a and Dmnt3b, referred to as the de novo Dmnts, are responsible for transferring methyl groups to unmodified DNA. In contrast, Dmnt1 copies the DNA methylation pattern from the parental strand to the newly synthesized daughter strand [15]. In essence, Dmnt3a and Dmnt3b directly participate in gene regulation, while Dmnt1 plays an essential role in the heritability and preservation of epigenetic changes.

The removal of methyl groups from cytosines can occur passively, as methylation can be lost during cell division without the involvement of any active mechanism. Active demethylation mechanisms, on the other hand, encompass a series of enzymatic reactions that convert cytosine to thymine, creating a guanosine-thymine mismatch that induces base excision repair machinery. Another active demethylation mechanism involves ten-eleven translocation (Tet) enzymes to modify 5mC to 5-hydroxymethyl-cytosine (5hmC), which can be converted back to cytosine through various pathways [15,16].

Repressing gene expression: Readers

DNA methylation can repress gene expression by hindering the binding of transcription factors to gene promoters. Conversely, certain proteins can recognize methylated sequences within the genome and bind to these sites, effectively blocking the binding of transcription factors. These proteins are categorized into three distinct families: the methyl-CpG-binding domain (MBD) proteins, the ubiquitin-like, containing PHD and RING finger domain (UHRF) proteins, and the zinc-finger proteins [15].

Histone modifications

When fully stretched out, the nuclear DNA from a single human cell spans approximately 2 meters [17]. Multiply that by the total number of cells in a human body, and you will obtain a number that could span astronomical distances. To accommodate this genetic material in a cell’s nucleus, it must be intricately coiled and densely packed — a feat achieved through the essential role of histones.

What are histones?

Histones are basic structural proteins present in the nuclei of eucaryotic cells, characterized by their richness in lysine and arginine residues. DNA wraps around histones much like a thread around a spool, forming structures known as nucleosomes. These nucleosomes, in turn, are bundled into 30-nm fibers, creating tightly compacted chromatin. There are five major histone families: H1, H2A, H2B, H3, and H4, each comprised of a globular domain and a tail domain [18,19].

As mentioned earlier, nucleosomes consist of a histone core encircled by DNA. The histone core is formed by a histone octamer comprising two H2A and H2B dimers and a H3 and H4 tetramer, around which 146 base pairs of DNA are tightly wound. Emerging from the core are the tail domains of the histones, which can undergo post-translational modifications to regulate important cellular processes such as DNA repair, mitosis, or meiosis, among others [20].

Histone functions

Histones serve a multifaceted role in safeguarding DNA from entanglement, protecting it against DNA damage, and contributing significantly to DNA replication. Given our focus on epigenetics in this article, we will hone in on their pivotal role in regulating gene expression.

The tail domains of histones, which protrude from the nucleosome, undergo post-translational covalent modifications at various amino acid residues. These modifications encompass methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, citrullination, and ADP-ribosylation. Additionally, the cores of histones H2A and H2B can also undergo modifications. Various combinations of these modifications, known as histone marks, are believed to form a code referred to as the histone code [21,22]. Importantly, all these modifications are reversible and they can be removed by specific enzymes.

These histone marks exert an impact on the conformation of histone tails and their interactions with DNA, as well as other proteins like transcription factors. For example, they can modulate DNA’s affinity for histones, thereby rendering specific regions of the genome more or less accessible to transcription factors. This, in turn, results in the regulation of gene expression [23].

Non-coding RNAs

RNA is a nucleic acid that serves as a bridge between DNA and proteins. Messenger RNA (mRNA) is transcribed from DNA within the nucleus and subsequently translated into proteins by ribosomes in the cytoplasm. Beyond its role as the intermediary molecule carrying genetic code, RNA molecules have a broad spectrum of functions, including structural contributions (for example, as a constituting element of ribosomes) and regulatory functions.

For example, microRNAs (miRNAs), which are 17-25 nucleotides long non-coding RNAs, have been shown to downregulate mRNA, thereby reducing the expression of various proteins encoded by those mRNAs [24,25]. Additionally, some mRNAs can directly or indirectly modulate the transcription of their own genes or other genes by regulating transcription factors. Other mechanisms of gene expression regulation by RNA molecules include alternative forms of splicing or the formation of double-stranded RNAs that interfere with mRNA transcription [26].

The Impact of Epigenetics

So far, we have briefly explored a few epigenetic mechanisms regulating gene expression in human cells. All of these mechanisms meet the criteria we outlined earlier: they influence gene expression without altering DNA sequence, they are reversible (methylation and histone modifications can be removed), and they are inheritable, being passed down to daughter cells after cell division.

However, the heritability of epigenetic changes extends beyond just the cells within an organism. A growing body of evidence suggests that these changes can also be transmitted across generations. For instance, non-coding RNAs can be transferred to the embryo by eggs and spermatozoids, and the potential transfer of histone marks and DNA methylation patterns remains a subject of debate [27].

Recent advancements in epigenetics have shed light on how gene expression is regulated, offering profound insights into cellular differentiation and stability maintenance. They also provide crucial insights into how cells adapt to a changing environment and respond to various challenges. Epigenetic changes are reversible, enabling cells to adjust their transcription profiles to better suit specific circumstances, such as nutrient deprivation, oxidative stress, or cell division, to name a few [10].

Given the pivotal role of epigenetic mechanisms in gene expression, alterations in these mechanisms can profoundly impact human health and contribute to disease. High-throughput analyses of DNA methylation patterns have revealed significant correlations with age markers across tissues. Importantly, this “epigenetic clock” is most highly correlated with biological age [28,29]. Moreover, epigenetic age is accelerated by a high body mass index and decelerated by a healthy lifestyle and diet [30].

On the other hand, mutations in genes encoding for epigenetic effectors, such as methylases and demethylases or histones, have been implicated in some human cancers [31]. Epigenetic regulation of gene expression constitutes a highly intricate network that is not yet fully understood. However, unveiling the functioning of these mechanisms promises invaluable insights into fundamental biological processes and holds the potential to pave the way for the development of new therapies for treating human diseases.

Epigenetics in Health Optimization Medicine and Practice (HOMe/HOPe)

The Epigenetics module of the HOMe/HOPe Essential Certification takes a deep dive into the three major epigenetic modifications above and teaches the practitioner a clinical approach to detecting and correcting imbalance throughout the five lessons.

These lessons include: Epigenetics: A Molecular Framework for HOMe/HOPe, DNA Methylation, Correction of Methylation Imbalances, Epigenetic Modifications: Histones and Non-Coding RNA, and Epigenomic Effects of Diet, Aging, and Lifestyle. This last lesson also includes an in-depth discussion on epi-nutrients, dietary sources of methylations, and practical applications of epigenetic aging clocks and the loss of methylation as we age.

Teach your clients how to modulate their epigenome to enhance both their healthspan and their lifespan. Because, as Dr. Ted, the founder of HOMe/HOPe, likes to say, genes are not your destiny.

Check out the Epigenetics module in the HOMe/HOPe Essential Course here.

Written by Ferran Riaño-Canalias, PhD

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