How Epigenetic Changes control our Genes

Written by Emily Locke

These topics await you:

1) One of the Foundations of Epigenetics: Histone Modifications

2) ChIP – a Significant Method in Epigenetic Research

3) Overview of well-known (and less well-known) PTMs

4) First-in-class Antibodies for Epigenetic Studies (Pan, H3, H4 and H2A)

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"Green tea helps against cancer!" - It has been known for some time that green tea is so healthy that it even improves cancer statistics in Japan [1]. But why is that? The answer to this question could only be found through epigenetics. The term "epigenetics", composed of the words genetics and epigenesis, i.e. the development of a living being, describes a field of research that deals with the influence of the environment on our genes [2]. So the question is under what circumstances a gene is switched on and when it is inactivated again. These changes in the activity of a gene are not based on a change in the DNA sequence, such as through mutations or recombination. Rather, they are based on chemical changes to the chromatin or to the proteins that bind to the DNA. Although these epigenetic imprints cannot be detected in the genotype, since the DNA sequence is not changed, they can still very well be observed in the phenotype and can also be passed on to daughter cells [3].

In the case of green tea, it is a specific epigenetic mechanism that triggers the anti-cancer effect: when the non-fermented tea leaves are brewed, the substance epigallocatechin-3-gallate (EGCG) is released. The EGCG then reactivates a gene that codes for a cancer-fighting protein. This gene is often methylated, especially in older people, and is therefore silent. Hence, when green tea is consumed, the gene is switched on again and can thus exert its cancer-fighting effect [2]. This is just one of many examples from epigenetics that show how environmental influences can regulate our genes. In the eyes of many scientists, this emerging field of research promises a better understanding of diseases and new knowledge about their treatment.

One of the Foundations of Epigenetics: Histone Modifications

One of the basic epigenetic mechanisms is the modification of so-called histones. These are basic proteins which, as a component of chromatin, are essential for the packaging of DNA. Two copies each of histones H2A, H2B, H3 and H4 form a protein complex of eight histones. This histone octamer forms the core of a nucleosome around which the DNA is wrapped. Such a nucleosome represents the smallest packaging unit of DNA [4]. From it protrude the ends of histone strands, which are the target of histone-modifying enzymes (see Fig. 1). The first descriptions of post-translationally modified histones date back to the 1960s. At that time, analysis of histones from calf thymus had detected methylated lysines and a short time later, acetylated lysines could also be detected [5, 6]. Today, methylation and acetylation of lysines represent the best-known histone modifications. However, many other post-translational modifications (PTMs) have been found on histones that play an important role in the regulation of gene expression (see Fig. 1).

Histone modifications can occur both at the unstructured N- and C-terminal ends of histone proteins and in the globular region within the nucleosome core (Fig. 1). A specific nomenclature has evolved to describe the different modifications: First, the name of the histone is given (e.g. H3). This is followed by the amino acid involved in its one-letter code (e.g. K for lysine) with the position of the amino acid in the protein. Now the type of modification is mentioned (e.g. Me for methyl, P for phosphate or Ac for acetyl). Finally, the number of methyl groups (in the case of lysines and arginines) can also be specified [7]. For example, the designation H3K4me3 stands for a trimethylation of the lysine at position 4 of histone 3.

Figure 1: Post-translational modifications of histones H2A, H2B, H3 and H4. Shown are the four major PTMs methylation, acetylation, ubiquitination, and phosphorylation. They occur at the N- and C-terminal ends of histone proteins as well as in the globular region within the nucleosome core [8].

ChIP – a Significant Method in Epigenetic Research

Before we take a closer look at individual post-translational histone modifications and their effects on gene expression, we will discuss one of the fundamental methods of epigenetic research in more detail: chromatin immunoprecipitation (ChIP). This method can be used to analyze the interactions of proteins with specific DNA segments in intact cells [9]. The aim is to find out whether the examined proteins are associated with specific gene regions [10]. ChIP can be used to investigate the binding of transcription factors to promoters, but also to explore the distribution of various histone modifications and thus the basis of epigenetic gene regulation [11].

The basic principle is to capture the protein-DNA binding that exists at one point in time by fixing it with formaldehyde (Fig. 2). Subsequently, the DNA extracted from the cells is fragmented into fragments of 50 to 1000 base pairs by sonication, leaving the bound proteins attached to the DNA. In the next step, the DNA pieces associated with the protein of interest are selectively extracted using an antibody specific for the DNA-associated protein. Subsequently, the isolated DNA-protein complexes are dissolved again by heat treatment (Fig. 2). The free DNA fragments can now be purified and, using further methods (e.g. PCR, NGS), can be quantified as well as identified. From this, it can be concluded whether or how strongly the protein was associated with the relevant DNA segment in the living cell [12].

Figure 2: Chromatin immunoprecipitation (ChIP) workflow. First, the binding between the DNA and associated proteins is fixed. Then, the DNA is cut into 50 to 1000 bp long fragments by ultrasonication. Using an antibody specific for the DNA-associated target protein, the DNA-protein complexes are selectively immunoprecipitated. After separation of the isolated complex, the DNA can be purified and its sequence determined [13].

The antibodies used in ChIP must be of high quality for the experiment to be successful. Our supplier Assay Genie offers a wide range of premium ChIP antibodies validated for both common and novel PTMs. These include well-known modifications such as methylation or acetylation as well as quite exotic ones such as neddylation or SUMOylation [14].

Methylation

Methylation represents the counterpart to acetylation and is the best-known epigenetic signal. In addition to histones, DNA can also be directly methylated. In this process, methyl groups are added to a cytosine nucleotide within a cytosine-guanine sequence (CpG) by DNA methyltransferases [14]. Freshly methylated CpGs lead to the recruitment of so-called repressor proteins, which inhibit the interaction between DNA and transcription factors. As a result, gene expression in the respective genomic segment is suppressed. In the case of methylated histones, the histone conformation is closed, so that transcription of the genes is no longer possible [3]. Histone methylation is found on both lysines and arginines [7].  

Phosphorylation

Phosphorylation describes a process in which a phosphate group of ATP is added to a molecule by a protein kinase. The reverse reaction, called dephosphorylation, is carried out by protein phosphatases. Histone phosphorylation can occur on amino acids with a hydroxyl group (serine, threonine, and tyrosine). The insertion of a phosphate group significantly increases the negative charge of the histone, which alters the structure of chromatin. Histone phosphorylation is diverse in function and can contribute to both activation and inactivation of gene expression [7].

Ubiquitination

Ubiquitination describes the attachment of ubiquitin, a protein consisting of 76 amino acids, to another protein. This marks the target protein for degradation by the proteasome or by lysosomes. Three main types of ubiquitination are known: mono- and polyubiquitination, and multisite monoubiquitination. While in monoubiquitination only one ubiquitin molecule is attached to the protein, in polyubiquitination multiple molecules are attached to a single protein. In multisite ubiquitination, more than one ubiquitin molecule is attached to a single lysine residue of the target protein [14]. Ubiquitination of histones is associated with activation of eukaryotic gene expression, but the molecular basis of this regulation is still largely unknown [15].

Beta-hydroxy-butyrylation

Having taken a look at four of the most important and well-known PTMs, we now turn to somewhat more "exotic" modifications. These do not enjoy great popularity, but nevertheless have interesting effects on the proteins involved. An example of such an exotic is beta-hydroxy-butyrylation. This is a process in which enzymes attach a ketone body, beta-hydroxybutyrate, to histones. This modification can affect the structure and thus the function of the affected histone proteins. Beta-hydroxy-butyrylation has been linked to a variety of histone-associated diseases and may therefore represent an exciting target for novel therapies [14].

SUMOylation

SUMOylation is a PTM in which so-called SUMO proteins bind covalently to lysine residues of other proteins. SUMO proteins (small ubiquitin-related modifier) have a structural similarity to ubiquitin and form a highly conserved protein family in all eukaryotes. SUMOylation plays an important role in many cellular processes, including protein-protein interaction, nucleocytoplasmic transport, signal transduction, and cell cycle regulation [16]. Protein stability can also be affected by this modification: SUMOylated proteins are often more stable than non-modified proteins [14].

Neddylation

Neddylation describes a PTM in which the ubiquitin-like protein NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is conjugated to the target protein. In this process, an isopeptide bond is formed between the carboxy group of the C-terminal glycine of NEDD8 and the Ɛ-amino group of a lysine in the target protein. The process is similar to ubiquitination, but other enzymes are involved. Neddylation plays a crucial role in various cellular processes, such as transcription, cell contacts, and regulation of the ubiquitin-proteasome system. Disregulation of neddylation may contribute to the development of various diseases, including cancer, neurodegenerative diseases, and heart disease [17].

Crotonylation

As a final PTM, let's take a brief look at crotonylation. In this recently discovered modification, a crotonyl-CoA molecule is attached to a lysine residue of the target protein. This is usually the Ɛ-amino group of lysines in histones. Crotonyl-CoA is the thioester between crotonic acid and coenzyme A. It is found as a metabolite in the degradation of the amino acids L-lysine and L-tryptophan. Crotonylation affects many different proteins, including histones, transcription factors and enzymes. It influences the function as well as the stability of the proteins. Depending on the localization of the crotonyl-CoA molecule, the activity of the target protein can be increased or decreased. This modification is also suspected to have an influence on the development of diseases such as cancer [18].

Here you will discover all ChIP-validated antibodies from Assay Genie:  ChIP-validated antibodies from Assay Genie 

First-in-class Antibodies for Epigenetic Studies

Epigenetic changes occur many times more frequently in the course of a lifetime than genetic mutations [19]. Many scientists are therefore certain that in the future, epigenetics will yield answers to some age-associated diseases that could previously not be explained genetically. Hence, epigenetic studies could offer impulses for innovative therapies and thus, provide new directions in medical research.

Fundamental to this is the analysis of epigenetic modifications, such as histone modifications. The post-translational modifications described here represent a small selection of the known PTMs. Would you like to learn more about other modifications? Then take a look at this page from our supplier Assay Genie! Here you will also find an overview of Assay Genie's comprehensive selection of highly validated antibodies for chromatin immunoprecipitation.

Our supplier Rockland Immunochemicals also provides you with high-quality tools for your epigenetic studies! For the detection of different histone modifications, the American company supports your research with a variety of specific antibodies, many of which can be used in chromatin immunoprecipitation. Also, get Rockland's "Histone Modifications Map" for your lab or office free with every online order!

Pan-specific

Histone H3

Histone H4

Histone H2A

Find all antibodies from Rockland that are applicable for ChIP:  ChIP-validated antibodies from Rockland 

Sources

[1] https://www.ruprecht.de/2019/11/12/gutes-fuer-die-gene/, last access: 11.01.2023

[2] https://www.planet-wissen.de/natur/forschung/epigenetik/index.html, last access: 11.01.2023

[3] https://de.wikipedia.org/wiki/Epigenetik, last access: 11.01.2023

[4] https://de.wikipedia.org/wiki/Histon, last access: 11.01.2023

[5] Allfrey, V. G., Faulkner, R., Mirsky, A. E. ACETYLATION AND METHYLATION OF HISTONES AND THEIR POSSIBLE ROLE IN THE REGULATION OF RNA SYNTHESIS. Proceedings of the National Academy of Sciences of the United States of America. 51(5):786-794 (1964).

[6] Murray, K. The Occurrence of Ɛ-N-Methyl Lysine in Histones. Biochemistry. 3, 10-15 (1964).

[7] https://de.wikipedia.org/wiki/Histonmodifikation, last access: 11.01.2023

[8] https://www.rockland.com/resources/histone-modifications/, last access: 11.01.2023

[9] https://www.assaygenie.com/chip-antibodies, last access: 11.01.2023

[10] Buck, MJ, Lieb, JD. ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. Genomics. 83(3):349-60 (2004). 

[11] Kimura, H. Histone modifications for human epigenome analysis. J Hum Genet. 58(7):439-45 (2013). 

[12] https://en.wikipedia.org/wiki/Chromatin_immunoprecipitation, last access: 11.01.2023

[13] https://commons.wikimedia.org/wiki/File:Chromatin_immunoprecipitation_sequencing.svg

[14] https://www.assaygenie.com/epigenetic-antibodies, last access: 11.01.2023

[15] https://gepris.dfg.de/gepris/projekt/5293400?context=projekt&task=showDetail&id=5293400&, last access: 11.01.2023

[16] Gareau, J., Lima, C. The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol. 11, 861–871 (2010). 

[17] Kandala S, Kim IM, Su H. Neddylation and deneddylation in cardiac biology. Am J Cardiovasc Dis. 4(4):140-58 (2014).

[18] Ntorla A, Burgoyne JR. The Regulation and Function of Histone Crotonylation. Front Cell Dev Biol. 9:624914 (2021).

[19] https://flexikon.doccheck.com/de/Epigenetik, last access: 11.01.2023

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