Epigenetics and Gene Expression

Evolution of Epigenetics

Epigenetics, predating its 20th-century naming, has been a critical evolutionary tool, enabling life to adapt and specialize through changing environments without altering genetic codes. This mechanism allowed early organisms to swiftly respond to challenges like climate shifts and nutrient scarcity. As life evolved from single-celled entities to complex multicellular organisms, epigenetics became something evolution capitalized more and more on. It began to play a pivotal role in differentiating diverse cell types from the same genetic material, ensuring the development and maintenance of specialized tissues and organs.

This article aims to explore the intricate role of epigenetics in controlling gene expression, highlighting its significance in the transition from simplicity to biological complexity. We start with the basics of gene expression control, setting the stage for a deeper understanding of epigenetic mechanisms.

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This article is part two of a four-part series on epigenetics.

1. Introduction to Epigenetics
2. Epigenetics and Gene Expression
3. Epigenetics and Aging
4. Coming Soon

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Gene expression control

Gene expression control is essentially how organisms adjust their functions in response to environmental changes. In its simplest form, it involves the control of the expression of one or more genes through the activity of proteins.. Although this falls outside the scope of epigenetics, due to the classical view that epigenetic changes must be heritable and involve mechanisms not altering the DNA sequence. Nonetheless evolutionarily this laid the foundations for epigenetics to exist. As life grew to be more complex and had to adapt to a wider variety of environments, evolution found the solution in epigenetics. Thus, understanding the basis, the starting point of epigenetics is important to understanding epigenetics as a whole. 

As life was taking its first steps towards becoming better at surviving it faced a problem. The primordial cells had to change their cellular states according to the changing conditions of the environment. A need to time the expression of some proteins arose. Some proteins that responded to the stressful environment these cells were in. Unstable conditions such as temperature, acidity, nutrient presence etc. pushed the cells to develop systems that allow the temporary activation of systems. The cells that were able to do this gained an evolutionary advantage over the ones that couldn’t. The example of heat shock proteins illustrates this point well. These proteins are quickly produced in response to stress from high temperatures, helping to stabilize other proteins that might otherwise fold improperly. This rapid response to stress highlights the foundational role of gene expression control in adapting to changing environments.¹

This capacity for adaptation, the ability to finely tune gene activity in response to external cues, marks the beginning of the journey toward epigenetic control. As time went on this primitive control of gene expression wasn’t enough. Life was too ambitious. Especially as multicellular life began to arise. The demand for control grew and evolution found solutions in epigenetics. And has again and again favored organisms that were able to build upon their epigenetic toolbox inventory. Now there are a wide variety of mechanisms epigenetics can rely on which we’ll go through in this article and their relationships with aging. 

Epigenetics Toolbox

Among the modifications leveraged by epigenetics to control expression, some directly target DNA, others target proteins that interact with DNA, and some even target the already expressed RNA. Most of these modifications are present in many organisms, but a key distinction between simpler and more complex organisms lies in how precisely they manage this genomic control. 

At the heart of epigenetics are DNA methylation and histone modifications, pivotal for their targeted effects on specific genes and genomic regions. These foundational modifications are executed by specialized enzymes, known as “writers,” “readers,” and “erasers.” Writers introduce these modifications, readers detect and interpret them to modulate gene expression, and erasers remove them. This dynamic interplay renders the epigenetic system highly adaptable, enabling organisms to respond to environmental stimuli effectively.

As we delve deeper into the epigenetic mechanisms, it’s crucial to understand this dynamic and reversible system of gene regulation. The subsequent discussion will explore how these foundational modifications—through their specific application by writers, nuanced interpretation by readers, and timely removal by erasers—contribute to the complex orchestration of gene expression across different organisms and environmental contexts. We’ll explore the biological complexities of epigenetics, paving the way to understand its significant impacts on health and disease.

DNA methylation

DNA methylation is perhaps the most basic epigenetic modification. It simply involves the attachment of a methyl group, which is composed of a single carbon atom and three hydrogen atoms to a base of DNA. Given its widespread presence across a variety of organisms, it is likely the first epigenetic modification to appear on the evolutionary timeline.² DNA methylation is found in a wide spread of organisms ranging from bacteria to mammals with some exceptions in the middle that are devoid of it.³ Although the exact mechanisms and purpose of DNA methylation between organisms can differ, the general concept remains the same. Perhaps the most primitive organism that has DNA methylation is bacteria. It commonly functions not for gene expression control but as a primitive immune system where the bacteria methylates its own DNA and uses it to differentiate it from foreign, invading DNA.⁴ There are enzymes that specifically only cut unmethylated DNA which protects the bacteria from virus invasions. Although not its primary use It can also function to control gene expression even in bacteria.⁵ For example, the SeqA protein in bacteria binds to hemimethylated DNA at the GATC sequence near the origin of replication. This binding inhibits replication initiation both by physically blocking the replication machinery and by repressing the synthesis of the DnaA protein, essential for initiating replication.⁶

As the complexity of organisms increases, so too does the variety and functionality of DNA methylation, particularly regarding the sites of methylation. In bacteria, the methylation of DNA primarily occurs at adenine bases within specific recognition sequences, such as the GATC sequence mentioned earlier. Moving up the evolutionary ladder to eukaryotes, including plants, fungi, and animals, the landscape of DNA methylation becomes more diverse and sophisticated, reflecting the increased complexity of these organisms.⁷ In fungi and plants, methylation can occur not only at adenine but also at cytosine bases. This cytosine methylation is particularly prevalent in the context of CpG dinucleotides, where a cytosine is followed by a guanine in the DNA sequence. However, plants exhibit a broader pattern of methylation, including methylation at CHG and CHH contexts (where H represents A, T, or C), which plays significant roles in the control of gene expression, suppression of transposable elements, and genomic imprinting.⁸ In animals, DNA methylation predominantly occurs at the cytosine bases within CpG dinucleotides, similar to fungi and plants. However, the distribution and functional implications of this methylation differ significantly. In mammals, for example, CpG methylation is widespread and is a key epigenetic mechanism involved in gene regulation, X-chromosome inactivation in females, genomic imprinting, and suppression of repetitive elements.⁹ Regions of high CpG density, known as CpG islands, are often found near gene promoters and can be differentially methylated to influence gene expression. In contrast to the primarily defensive role of methylation in bacteria, in mammals and other eukaryotes, DNA methylation serves more complex regulatory functions, including developmental regulation, differentiation, and response to environmental factors.

Figure 1: Simplified Schematic of DNA Methylation and Demethylation. Sourced from Valente et al.¹⁰

The enzymes that maintain in “write” DNA methylation are called DNA methyltransferases (DNMTs). There are more than one variant found in mammals that serve to control methylation in different contexts. These enzymes are required for appropriate development and differentiation. In mice when the DNMTs are knocked out (deleted) they result in significant growth retardation.¹¹ The extent of the detriments upon knockouts extend all the way to cell death in human embryonic stem cells.¹² The TET enzymes, short for Ten-Eleven Translocation methylcytosine dioxygenases, play a pivotal role in the dynamic regulation of DNA methylation, acting as the “erasers” in the epigenetic machinery. These enzymes facilitate the removal of methyl groups from methylated cytosines, a process crucial for active DNA demethylation. The TET enzymes have an even more severe result upon knockout in mice. TET deficient embryos exhibit developmental arrest at the 2 cell stage.¹³ These studies go to show that DNA methylation or demethylation alone isn’t what’s explicitly important but the dynamic interplay between the DNMTs and the TET enzymes, which remove them. This allows for a finely tuned regulation of gene expression, crucial for normal development, cellular differentiation, and the maintenance of genomic integrity.

Aberrant DNA methylation is seen in diseases and in aging. As organisms age, their DNA methylation patterns change significantly, impacting gene activity, cell function, and susceptibility to diseases.¹⁴ These methylation changes, common in aging across various species, including humans, typically involve a reduction in DNA methylation, especially at CpG islands.¹⁵ This reduction can activate genes or elements that were previously inactive, possibly leading to genomic instability and a higher risk of cancer in the elderly. On the other hand, some genome regions might see an increase in methylation, causing the suppression of genes critical for cell protection and repair. This can increase the risk of age-related diseases, such as neurodegenerative and cardiovascular diseases. In fact the DNA methylation changes that happen with age were so significant that scientists were able to develop “DNA methylation epigenetic clocks” that serve as biomarkers that can predict an individual’s biological age.¹⁶ These clocks now stand as promising biomarkers for assessing biological age, increasingly utilized in scientific studies to understand and potentially mitigate the effects of aging. In the next article, we will dive deeply into the science behind these epigenetic clocks, exploring how they are constructed and their implications for research and health. 

Histones and their modifications

Histone modifications arguably represent the most significant epigenetic mechanism influencing gene expression. Histones are essential for the primary level of genome organization in the nucleus, forming the basis for the epigenome’s existence. The genetic material is incredibly long, and simply expressing its length in numbers of base pairs doesn’t do it justice. Let’s use a more tangible example. In each human cell, almost 6 billion base pairs of DNA exist.¹⁷ Each DNA nucleotide measures exactly 0.338 nanometers.¹⁸ Multiplying that by the number of bases in each human cell results in a total length of over 2 meters (6.5 feet), likely taller than you are. This length, over 260,000 times larger than the average nucleus width of 7.5 micrometers, packs into the nucleus 30 trillion times to form you.¹⁹ For comparison, a spool of floss with a diameter of around 10 cm and a length of 50 meters compacts only 5000 fold, 52 times less. And biology achieves this while maintaining regular and organized expression of genomic locations relevant to each cell type. Evolution has solved this problem with histones. But what exactly are they?

Figure 2: Chromatin Structure Overview. Sourced from NIH genome.gov²⁰

Proteins called histones, formed from multiple subunits, act as spools around which DNA winds. At the core of a histone’s structure, four core protein types are quintessential for forming a histone core. These proteins pair up, resulting in a histone octamer (a protein complex composed of 8 subunits), around which DNA wraps to form the nucleosome—the fundamental unit of chromatin. While each of these protein types has alternatives, adding complexity and variability to histone composition and function, their primary arrangement provides the necessary framework for DNA compaction and epigenetic regulation. Histones don’t just serve as passive structures for DNA winding; they are dynamic. Various modifications along their “tails,” such as methylation, acetylation, phosphorylation, and ubiquitination, serve as intricate codes that regulate gene expression by altering chromatin structure or recruiting transcriptional machinery to their genomic site.²¹ The sheer number of modifications histones can go under is illustrated by figure 2. We’ll obviously not talk about all of them. But just keep in mind that all of these are just one aspect of the immense complexity of epigenetics.

Figure 3: Histone Modifications Overview: This figure illustrates the various ways histone proteins (components of chromatin) can be modified, such as through adding chemical groups like acetylation or methylation. Sourced from Yang et al.²²

The interplay of these modifications forms a complex regulatory network, effectively modulating DNA accessibility to the cellular machinery responsible for reading genetic information. We have identified 311 proteins involved in writing, reading, and erasing histone methylation and acetylation alone in humans, with probably others yet to be identified.²³ The scale and complexity of control that epigenetics exerts through histones are hard to comprehend and significantly dwarfs the capabilities of other epigenetic mechanisms. This is also partly because histones have received the most research attention in the field of epigenetics, save for DNA methylation.

Among the many histone modifications that exist we’ll mention the (arguably) most two. Acetylation of histone tails commonly associated with transcriptional activation.²⁴ Enzymes known as histone acetyltransferases (HATs) add acetyl groups, usually reducing the positive charge on histones and diminishing their interaction with negatively charged DNA. This loosening allows transcription factors and other proteins easier access to specific gene regions, promoting gene expression. Conversely, histone deacetylases (HDACs) remove these acetyl groups, generally condensing the DNA and silencing gene expression. On the other hand histone methylation is more nuanced. Histone methylation can activate or repress gene expression, depending on which amino acids in the histone tails get modified and how many methyl groups are added. For example, triple methylation of 4th lysine amino acid on the tail of histone 3 (H3K4me3) is an active chromatin mark whereas the triple methylation of the 9th lysine (H3K9me3) is a repressive one. Histone methyltransferases (HMTs) carry out these modifications, and histone demethylases (HDMs) can reverse them.²⁵ The specificity and reversibility of histone modifications make them versatile tools for dynamic gene regulation. These modifications are not just about turning genes on or off; they also fine-tune gene expression levels and coordinate the expression of gene networks.

Chromatin, the complex of DNA wrapped around histones, organizes into two distinct categories: euchromatin and heterochromatin. Euchromatin, characterized by its loosely packed structure, serves as the stage for active gene transcription, allowing easy access for the transcriptional machinery. In contrast, heterochromatin, with its tightly coiled configuration, silences gene activity, maintaining genomic integrity and regulating gene expression through its compactness. As organisms age, a notable phenomenon is the gradual erosion of heterochromatin.²⁶ This degradation disrupts the usual silence within these regions, leading to aberrant gene expression patterns not typically observed in a given cell type. This aberrant expression is a consistent occurrence during aging. There are specific histone marks that change and some cell types that are disproportionately affected by this erosion such as stem cells. We’ll get into the details and causes of this erosion in the next article.

Non-coding RNAs (nc-RNA) and their modifications

Delving deeper into the epigenetic toolbox, we venture into increasingly unfamiliar territory. Histone modifications and DNA methylation are the common epigenetic mechanisms we know of today. In the previous article we’ve talked about the ~20,000  protein coding genes present in the genome and how they are controlled. But if you’re somewhat unfamiliar with the field of biology it’ll probably shock you to learn that the genes that express the  entirety of the proteins in a cell only account for a mere 1-2% of the genome.²⁷ Despite this protein-coding genes have long dominated the landscape of genetic research. This focus has overshadowed a vast expanse of the genome, once dubbed “junk DNA,” which we now understand plays a critical role in regulating these genes. Among the findings unearthed from this non-coding majority are non-coding RNAs (ncRNAs), a group of molecules that, despite not translating into proteins, wield immense influence over cellular processes and genetic regulation.²⁸

NcRNAs are categorized by their size into long non-coding RNAs (lncRNAs) and short non-coding RNAs (sncRNAs), with 200 base pairs serving as the dividing line. LncRNAs, exceeding this length, engage in various cellular functions including chromatin remodeling and multi-level gene regulation.²⁹ They operate through diverse mechanisms, such as sequestering proteins from their intended targets or directing protein complexes to specific DNA regions, thereby modulating gene expression. For example LncRNAs have been shown to promote an open chromatin structure by neutralizing the positive charge of histones through their own negative charge.³⁰  SncRNAs on the other hand, are typically shorter than 200 nucleotides and encompass subclasses like microRNAs (miRNAs) and small interfering RNAs (siRNAs). MiRNAs regulate gene expression post-transcriptionally, targeting messenger RNA (mRNA) for commonly degradation / translational repression but sometimes even activation, thereby fine-tuning protein synthesis essential for development, apoptosis, and other cellular processes.³¹ The transcription control of miRNAs have been leveraged in processes like cellular reprogramming. With the expression of pluripotency-related miRNAs the efficiency of reprogramming was increased to 90%.³² The other type of sncRNA, SiRNAs partake in the RNA interference (RNAi) pathway, targeting mRNAs for destruction to prevent protein production, playing roles in gene silencing and antiviral defense.³³

As we explore the implications of ncRNAs in the context of aging, their significance becomes increasingly apparent. Changes in ncRNA expression and function have been linked to the aging process, suggesting their involvement in the regulatory networks that influence cellular senescence and longevity. For instance, alterations in miRNA profiles have been associated with age-related diseases, modulating pathways involved in cellular aging such as stress response and DNA repair.^{34 35} In fact the levels of certain age related miRNAs were shown to decrease upon heterochronic parabiosis, a procedure where old mice are exposed to the circulation of young mice leading to rejuvenation in the old.³⁶ LncRNAs, through their intricate interactions with chromatin and protein complexes, they contribute to the epigenetic alterations observed in aging cells, affecting processes such as genomic stability and inflammation.³⁷

The world of ncRNAs is vast and still not very well understood. Understanding the roles of ncRNAs in aging not only enhances our grasp of the fundamental processes underlying cellular senescence but also opens up new avenues for potential interventions aimed at mitigating the effects of aging and improving healthspan. The exploration of ncRNAs in the context of epigenetics and aging represents a frontier with promising implications for biology and medicine, offering insights into the mechanisms that govern the transition from vitality to senescence. Who knows in the future we might even leverage their abilities for aging interventions.

3D chromatin structure

The final mechanism that we’ll talk about in the world of epigenetic regulation is 3D chromatin organization—a facet of genetic control that, while critical, is still being unraveled by scientists. This aspect of epigenetics examines how chromatin’s physical arrangement within the nucleus influences gene expression and cellular function. Chromatin isn’t randomly dispersed within the nucleus. Instead, it adopts specific structures and formations that facilitate or hinder the access of transcription machinery to certain genes. This spatial organization is crucial for regulating the timing and level of gene expression, impacting everything from cell differentiation to the aging process.³⁸

Figure 4: Nucleus and Chromosome Layout: This figure shows chromosomes in the nucleus, each in its own space, and how active and inactive DNA areas group together. It explains how DNA folds and organizes, affecting which genes are turned on or off. Sourced from Zheng et al.

Within the interphase nucleus, individual chromosomes establish distinct territories, revealing a structured landscape of functional zones. Active genes prefer the company of nuclear speckles in “compartment A,” where they engage with other active regions across different chromosomes, enhancing their expression. Meanwhile, inactive genes align with the nuclear lamina or nucleolus in “compartment B,” preferring isolation that correlates with their silenced state.^{39 40} This spatial segregation ensures efficient gene regulation, with active genes positioned for optimal expression and inactive ones maintained in a dormant state.

Central to the genome’s structural organization are topologically associating domains (TADs), which act as genomic neighborhoods where genes and their regulatory elements cluster together for coordinated expression.⁴¹ Architectural proteins, including CTCF and the cohesin complex, along with cohesin-loading factors NIPBL and MAU2, play pivotal roles in sculpting chromatin domains. CTCF and cohesin work together to form loops that segregate and organize chromatin, while NIPBL and MAU2 are essential for loading cohesin onto the DNA, facilitating the looping process.⁴² Their actions dictate the folding patterns of chromatin, essential for gene regulation, differentiation, DNA replication, and repair. This looping mechanism, influenced by the encounter between cohesin and CTCF, underscores the genome’s dynamic and functional architecture.³⁸

Alterations in 3D chromatin organization have been linked to various diseases, including cancer, where aberrant gene expression due to changes in chromatin structure can drive the development and progression of tumors.⁴³ Similarly, disruptions in chromatin organization are associated with aging, where changes in chromatin structure can influence the expression of age-related genes and contribute to the decline in cellular function.^{44 45}

The epigenome in a single picture

Having delved into the fundamentals of the epigenome, it must be kept in mind that this article only scratches the surface of a profoundly complex field. Not only are there aspects of epigenetic regulation that remain unexplored within this article, but the scientific community continues to uncover layers of complexity that were previously unknown. However with each new discovery we learn again that they play into the coordinated orchestra that is the epigenome. None of the epigenetic modifications should not be considered in isolation. For instance, a specific chromatin scenario that exemplifies the collaboration and coordination of epigenetic mechanisms talked about in this article all working in concert. Such a scenario might involve a gene critical for cell differentiation, where DNA methylation silences competing genetic pathways, histone modifications adjust the chromatin’s accessibility, nc-RNAs guide or inhibit transcription factors, and the 3D chromatin organization brings distant regulatory elements into close proximity with the gene. This integrated approach underscores the epigenome’s role as a dynamic, complex system, where its components coalesce to fine-tune gene activity with precision.

A unifying theme across these mechanisms is how they change with age; they all tend to erode, leading to a loss of epigenetic information. This degradation manifests in various specific ways, fundamentally altering gene expression patterns that are crucial for maintaining cellular function and organismal vitality or by initiating expression of genes that are supposed to be silenced in that cell type. As these epigenetic landscapes shift, so too does the biological basis of longevity and disease. The next articles will delve deeper into the hows and whys of epigenetic deregulation during aging, examining the specific processes by which these mechanisms falter and exploring the potential interventions that could mitigate or even reverse their detrimental effects. Understanding these pathways in greater detail will not only illuminate the complexities of aging but also pave the way for novel strategies in the pursuit of longevity and the treatment of age-related conditions.

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