What is epigenetics?
Epi is a Greek word for ‘above’, which implies that epigenetics is something on the top of our traditional understanding of inheritance.
In the past, scientists were able to observe a lot of events that could not be explained by genetic knowledge. Conrad Waddington, a pioneer in the field, defined epigenetics in the 1950s as changes to the genes expression induced by the environment. Quickly, numerous unexplainable biological phenomena were coined as ‘epigenetic’ changes, while the molecular mechanisms behind them remained largely unknown.
Identical twins have exactly the same genetic code as they originate from one zygote, which is then separated into two distinct embryos. What’s more, they’re subjected to identical conditions during key, early stages of development in the mother’s womb, and, unless they’re separated after birth, they are also brought up in similar environments. Given all that, it’s very surprising that the risk of developing highly heritable conditions like schizophrenia among identical twins (if one sibling already suffers from it) is only 50%. If DNA sequence was all that matters, then identical twins would always be identical in every possible aspect.
However, it turns out that not only the script is of importance, but also the instructions on how to properly interpret it.
Molecular mechanisms
These phenomena of genetically identical organisms actually appearing very different to each other need to have some molecular explanation. This led to seeing epigenetics as modifications to the genetic material that change the genes being expressed, in other words, switched on or off, but which don’t alter the genes themselves. To be fair, it’s easy to say that all the unexplainable changes are due to the influence of the ‘environment’.What really matters, is figuring out how the environment actually does that. There must be a way in which various stimuli physically affect our gene expression machinery, even though the genetic sequence itself remains intact.
Two of the most important mechanisms that regulate gene expression are DNA methylation and histone modifications. The necessary condition for that is the accessibility of DNA, which occurs when it’s not tightly coiled as chromosomes. DNA methylation is a gene-silencing modification, thereby it causes the formation of compact chromatin. It takes place when a methyl group is added to one of the nucleotides of DNA, cytosine, by the group of enzymes called DNA methyltransferases. Importantly, this process occurs almost exclusively for cytosines that are placed next to guanines, forming so-called CpG islands. The methylation of DNA sometimes may physically disrupt the binding of transcriptional proteins to the gene, but it’s far more likely that methylated DNA is bound by proteins known as methyl-CpG-binding domain proteins (MBDs), which then further recruit additional chromatin remodelling proteins that modify histones, thus forming inactive chromatin (heterochromatin).
In almost every organism analyzed, it was found that if the methylation is located in a gene promoter, it acts to repress gene transcription. CpG-dense promoters of actively transcribed genes are never methylated, but, conversely, silent genes do not necessarily have to be methylated. In total, 60–70% of genes have a CpG island in their promoter region and most of these CpG islands remain unmethylated independently of the transcriptional activity of the gene.
The second major epigenetic mechanism is histone modification, specifically acetylation and deacetylation. The octamer of histone proteins with a DNA tightly wrapped around them makes up a structure called a nucleosome, which are then coiled into chromosomes. Looking more specifically at histone conformations, all of them are made of alpha-helical structures and N-terminal tail, which is variable among histone types. This is precisely where all epigenetic modifications are occurring. The most common one is acetylation, which means adding an acetyl group on lysine or arginine residues in the tail. These positively charged amino acids attract negatively charged DNA, making the nucleosome coiling tight. Upon acetylation, lysine and arginine become neutral, so that there’s no longer any attractive electrostatic attraction and the DNA becomes loose. This energetically demanding reaction requires an enzyme to proceed- a histone acetyl transferase. It was observed that acetylated histones contribute to the formation of euchromatin, an easily accessible DNA that is ready to be transcribed and expressed. On the other hand, deacetylation performed by histone deacetylase favours tight coiling of nucleosomes, forming inaccessible heterochromatin.
Two other frequently observed types of modifications are methylation (adding methyl group on lysine or arginine) or phosphorylation (adding a phosphate on serine or threonine). With methylation, the nature of the epigenetic modification depends on which residue is methylated and what type of methylation is it, so it can both have an activating and repressing effect. As for the phosphorylation, it is mostly considered as inducing increased gene expression, as negatively charged phosphates added to the histone tails repel negatively charged DNA backbone, resulting in a DNA strand being packed more loosely.
Gene expression control
Gene expression describes the production of a functional product, such as proteins, from a series of nucleotides in DNA, by transcription of a gene into RNA. Every cell in the body, except for the gametes which contain one copy of each chromosome rather than two, contains all the genetic information of the organism. However, as the cells become specialized into different cell types (for example, a red blood cell, a neuron, a hepatocyte in the liver, a muscle cell), they express different genes.
But how can a cell know which genes to express and which genes to silence to serve its purpose? This process is known as the regulation of gene expression. Imagine what would happen if there was no regulation of gene expression – your eye cells would express the same genes as your stomach cells and, therefore, start secreting hydrochloric acid!
However, genes cannot control an organism on their own, but they will rather interact with the organism’s environment. Some genes are always expressed regardless of the environmental condition. They are called constitutive genes and control the fundamental processes within an organism: DNA replication, transcription, repair, central metabolism and protein synthesis are some examples. The other type of genes are regulated genes whose expression gets turned “on” and “off”, or “up” and “down” (like a rheostat), depending on the environmental conditions.
In prokaryotes, genes are regulated in a less evolved manner by classical mechanisms involving activator and repressor proteins binding to DNA. Most of the regulatory proteins are negative so they turn “off” gene expression. For example, for the formation of tryptophan in E. coli, three enzymes are required. It takes a total of five genes that are very close to one another on the bacterial chromosome, and one promoter to synthesise the needed enzymes. In this case, there is a segment of DNA between the promoter and the first of the five genes, called an operator which acts as an on/off switch. The operator controls whether the RNA polymerase has access to transcribe the downstream genes. This whole system made by the promoter, the operator, and the genes are called an operon. Normally, the operon is “on”, but if a specific repressor binds to the operator, then the promoter is blocked, and RNA polymerase cannot start transcribing. Thus, the repressor inhibits gene expression and stops the production of tryptophan when, for example, there is enough tryptophan in the environment. E. coli only needs to make tryptophan when environmental levels of tryptophan are low, and the operon would be switched “on” in this case. However, there are also genes that are typically “off” and need to be activated. In E. coli, there are genes that produce an enzyme responsible for breaking down lactose into glucose and galactose. Normally, there is a repressor bound to the operator preceding these genes, but an isomer of lactose can deactivate the repressor, thus allowing for transcription of the genes and higher levels of metabolized lactose. These genes are turned “on” when environmental levels of lactose are high. These mechanisms are examples of negative gene regulation. This is not an epigenetic mechanism as it does not involve chemical changes to the DNA and bacteria do not have histone proteins, although it allows the bacteria to respond to environmental changes.
Eukaryotes also regulate genes using transcriptional activators and repressors. However, histone modification and DNA methylation are additional methods of regulating gene expression in eukaryotes. There are some very well-known examples of epigenetics used to regulate the expression of genes.
Genomic imprinting is one example of gene regulation by epigenetic mechanisms. In this process, one of the two alleles of a gene is silenced for the entire life span of the cell, depending upon the sex of the parent from whom the gene was inherited. Some genes are silenced when inherited from the mother, and others when inherited from the father. Genomic imprinting results from methylation of the DNA and histone modification, and when it is combined with a genetic mutation, it can lead to disease. For example, when deletion of a particular DNA sequence in the maternal chromosome 15 occurs, this leads to Prader-Willi syndrome in the child. When the same sequence is deleted from the paternal chromosome 15, the child will have Angelman syndrome.
One other mechanism involving epigenetic gene silencing for the entire lifespan of the cell is X chromosome inactivation. One X chromosome has over 1000 genes that ensure cell development and viability, but as females carry two copies of the X chromosome, there is a risk of toxic double-dose of X-linked genes. To avoid this risk, female mammals have found a way to shut off one of the two copies of chromosome X by transcriptional silencing and then compact it into a stable structure called a Barr Body. This process involves transcription, the participation of two noncoding, complementary RNAs (XIST and TSIX) which initiate and control the process, and CTCF, a DNA-binding protein. Xist “coats” the X chromosome to be silenced, followed by epigenetic changes to that X chromosome. One well-known example of X-chromosome inactivation is the colour pattern of calico cats. The genes coding for fur pigmentation are X-linked and each X-chromosome will result in a different colour when left active (either orange or black). Most of the calico cats are females as X-chromosome inactivation mostly occurs in cells with multiple X-chromosomes. This inactivation process is widely researched in the field of cancer biology, as it was shown that the active state of both X-chromosomes is linked to human breast and ovarian tumour formation.
Epigenetic changes can also occur due to bad nutrition during pregnancy. We can take the example of babies whose mothers were pregnant during the Dutch Hunger Winter Famine between 1944 and 1945. When researchers looked at gene expression in those babies, 60 years after the famine, they discovered that those people had increased levels of methylation at some genes and decreased levels at others when compared to their siblings who were born under normal environmental conditions. These epigenetic changes can explain why humans born during harsh conditions (e.g. famine, war) are more likely to develop a disease such as a type 2 diabetes, heart disease and schizophrenia during their adult life.
Epigenetics in applications
- Medicine and therapies
Most illnesses, such as cancers, neurodegenerative disorders, cardiovascular diseases or any ageing-related conditions are often associated with environmentally influenced alterations, which can also be called epigenetic. Scientists were always looking to discover why certain people don’t respond well to standard therapies and drugs. Only after realising the importance of epigenetics, the concept of personalized medicine was born, promising a revolutionary approach in combining both genetic and epigenetic diagnostic testing. This would allow creating an individual’s personal genomic profile by discovering all relevant molecular alterations in cells: due to genetic heterogeneity and epigenetic ones.
In human cancer development, it’s very common that oncogenes, such as the MYC proto-oncogene, are epigenetically activated at some point. Furthermore, cancers frequently use epigenetic mechanisms to deactivate cellular antitumor systems by methylating genes called tumour-suppressors. Either of the two mechanisms (or two simultaneously) lead to a significant imbalance in the rate of cell division, proliferation and death, ultimately causing tumour progression and irreversible organ damages. With the development of various drugs targeting epigenetic regulators, epigenetic-targeted therapy has been applied in the treatment of haematological malignancies and has exhibited viable therapeutic potential for solid tumours in preclinical and clinical trials. Although epigenetic therapy has a rational and profound basis in theory, some problems remain to be discussed and solved. The most important problem is selectivity because epigenetic events are distributed across normal and cancer cells. Therefore, the priority is to determine the most important epigenetic alterations for different, specific types of cancers, so as to avoid targeting healthy cells.
Furthermore, a fuller understanding of the specific mechanisms underlying those alterations in different cancers is necessary to design safe and accessible therapies. Personalized medicine seems to be a great fit for increasing the specificity and safety of therapies for all patients, taking into account their individual, unique genomic and epigenomic data. It’s also worth noting that one of the most significant advantages of epigenetic therapies is that they are fully reversible, unlike gene therapy, which means that they potentially induce less risk.
- Drugs and ageing
Epigenetic modifications such as DNA methylation, histone modifications and alteration in microRNA expression, are highly reversible in normal tissue but can become imbalanced and inheritable in tumours or other abnormal cells. These epigenetic changes play an important role in controlling gene expression and genomic stability in the entire life of an organism. When epigenetic dysregulation occurs, there may be a causal effect on already existent age-associated diseases such as cancer, diabetes, and the decline in the immune response. Additionally, there is extensive evidence that epigenetic mechanisms are involved in synaptic plasticity and play a key role in memory and learning; therefore, any dysregulation of an epigenetic mechanism in the brain can lead to neurodegenerative or psychiatric diseases. Thus, one of the main scientific interests of today’s world is the so-called “epigenetic drugs” which act on the enzymes that are responsible for generating epigenetic modifications. One example is drugs based on the superfamily of histone deacetylases (HDACs) including HDAC 1-11 and sirtuins (SIRT) 1-7.
HDAC inhibitors are promising candidates in the treatment of cancer as they have shown anti-tumour activity against haematological malignancies; in the treatment of neurodegenerative diseases such as Huntington’s disease, Parkinson’s disease, Alzheimer’s disease and Rubinstein–Taybi syndrome (by ameliorating deficits in synaptic plasticity, cognition and stress-related behaviours); anxiety and mood disorders; and in the regulation of the innate immune response against microbial pathogens.
Both sirtuins inhibitors and activators have gained much scientific interest as a therapeutic approach for treating metabolic, cardiovascular and neurodegenerative diseases, and cancer. It was reported that some sirtuins inhibitors had anti-proliferative effects in cell cultures and mouse tumour models. Additionally, sirtuin inhibitors of SIRT2 and AGK-2 were reported to be effective for the treatment of Parkinson’s disease, while other sirtuin activators can have protective effects against Alzheimer’s disease.
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