Epigenetics is a research area that started to receive increasing attention at the end of the 1990s and took off during the last two decades. Today, it is accepted that epigenetic changes due to an unhealthy, sedentary lifestyle, obesity, stress, aging, and many other environmental factors will influence our health throughout life.

This is the reason why I added this chapter. You will only fully understand how nutrition and lifestyle contribute to health if you understand the big ideas of epigenetics.

Epigenetics is a complex molecular concept and at the moment we only understand the big ideas and a flurry of isolated mechanisms. Imagine the situation as a huge puzzle. We have an idea what the picture will most likely look like, but at the moment there are many disconnected islands of puzzle pieces.

It will take a lot of work by scientists to fully understand how obesity and lifestyle change the epigenome, and more importantly what we can do about it.

I still think it is important for a future health professional to understand the basics. Only that way you will understand the full impact of the obesity epidemic and why it is so important to turn the situation around.

Your biology course might be a while back. Here is an infographic reviewing the major concepts you need to understand to learn about epigenetics. I added the URL for the videos at the end of the chapter as well.

Something Didn’t Make Sense

1953 Watson and Crick published their paper “MOLECULAR STRUCTURE OF
NUCLEIC ACIDS A Structure for Deoxyribose Nucleic Acid”  describing the structure of the DNA for the first time. Their discovery was based on the research of many scientists and changed the understanding of the human body. Understanding the structure of DNA allowed us to start the process of understanding diseases with a genetic predisposition and scientists set out to identify genes coding for those diseases.

In 1990 the National Institute of Health (NIH) joined with international partners in a quest to identify and sequence the entire human genome. The genome project was a big deal not only in the scientific community—keep in mind at that time the internet was not publicly available—but also in public. Public relation efforts promoted the human genome project in the media. People were aware of it.

The big idea was: Once we know the sequence of the base pairs of all genes, scientists will have a powerful tool to identify where diseases are located on the genome. This will help with diagnosis, treatment and prevention of diseases. We will be able to predict who will develop a disease later in life and take preventative measures. Maybe we will get to the point when science can just repair those genes. In 2003 the scientist working on the project completed the Human Genome Project. Press conferences followed.

The sequence was made available to the world public and as a result many research groups worldwide were able to identify the genetic code (genotype) for common and rare diseases. Today there are more than 2000 genetic tests for human conditions.

The Human Genome Project finished identifying the DNA sequence by 2003 but something didn’t make sense.

You probably know several identical twins. Identical twin babies look so similar that parents often tie ribbons around their ankles to tell them apart.  As the twin babies grow, they start to look slightly different and the differences become more pronounced the older the twin pair gets. This is measurable as well: 30% of identical twins end up with a different height.

Scientists realized something similar. Twin studies are very popular in science with one twin being exposed to a treatment and the other twin being the control.

This setup doesn’t always work. In twin studies researchers found something surprising. Both twins might for example have the genetic marker for colon cancer or T2D —the genotype—but only one twin develops the disease during their lifetime—the phenotype. Something didn’t make sense here: If the DNA is the sole blueprint for our body then this shouldn’t happen. If both twins have a genetic disease marker both twins should develop the disease.

This was not the only riddle. Humans start out with a single cell, the zygote, that starts dividing. All daughter cells have the same set of DNA, the blueprint. This is called a totipotent cell.

During embryonal development cells differentiate though. All cells have a full set of genes, but something tells one cell to become a brain cell, another a skin cell, another a muscle cell, bone cell, liver cell and so forth. This “something” silences genes that are not needed for the function of the specific cell.

The Epigenome Regulates Which Genes Are Expressed and Which Ones Are Not

This ‘something’ is the epigenome. Epi is Greek and means above. In this case above the genome. The epigenome regulates which genes are expressed and which ones are not. This allows a stem cell, an omnipotent cell, to become and function for example as a brain cell.

Those epigenetic switches turning genes on and off are laid down during embryonal development. From then on, those switches are mitotically inheritable, meaning after cell division every daughter cell will have the same epigenetic switches (marks).

Epigenetic switches also explain the phenotype of a person, the observable characteristics such as skin and hair color, size, and biochemical properties. Just like the genome, most of the epigenome is inheritable.

The Epigenome Organizes The Genome by Wrapping the DNA Around Histones

If you want to understand how those epigenetic switches work, you will need to understand how DNA is organized. We tend to think of the genes as Xs, but genes appear in this form only during mitosis.

The DNA is also not a long string bunched up in the nucleus but is packaged in an elaborate way. This packaging allows to squish 6 feet of DNA into 6 µm of nucleus.

Here is a video explaining the packaging. Watch the video until 1:17. You can also copy and past this link into your browser: https://youtu.be/OjPcT1uUZiE.

The cell is using this wrapping method around histones to turn genes on or off. If the DNA is loosely wrapped around the histones the gene is switched on. It is accessible for the transcription machinery to copy and produce a strand of messenger RNA. Loosely wrapped, accessible genes are called euchromatin. Euchromatin has a beads on a string appearance as you see below (left side: loose purple beads on a yellow background).

If genes are wrapped tightly (picture above right side) genes are not accessible and turned off. This tight wrapping of DNA around the histones is called heterochromatin.

The question is, how can DNA be wrapped tightly or loosely. Watch the video starting at 5:10 min.

The epigenetic enzyme machinery can unwind the DNA from the histones and start the process of transcription. A tightly packaged gene (heterochromatin) cannot be unwound and is therefore turned off. This happens by adding specific epigenetic marks to the DNA or the tails of histones. The additional tags strike bonds with other histones, histone tails or DNA therefore tightening the package.

Epigenetic Marks Determine the Packaging and Can Be Attached to the DNA or Histone Tails

There are two types of epigenetic marks. Adding a methyl group directly to the DNA or attaching small functional groups to histone tails.

The first type of epigenetic marks are methyl groups attached to the DNA. The methyl groups are mostly attached to specific regions of the DNA, the CpG islands. CpG islands are rich in cytosine (C) and guanine (G) base pairs. The methyl group is added by an enzyme to the cytosine.

The second type of epigenetic mark is the attachment of a functional chemical group to a histone tail.

As we established, the DNA is wrapped around the histone octamer. You can envision the histones as protein bunches with amino acid strings sticking out like a tail. Enzymes can attach a variety of functional groups to those tails. Examples are methyl, acetyl or propionyl groups, but also the vitamin biotin. There are plenty of other groups used as epigenetic marks. Enzymes can just attach one mark or several at the same time. There are more than 50 different sites for epigenetic marks located on the histone tail.

You can imagine the huge amount of possible epigenetic tag combinations this allows. Today, only a few select sites or combinations are researched and much of the histone code is not identified.

We do know that histone tail methylation will often compact a gene turning that stretch of DNA into heterochromatin (switching off).

Acetylation of histone tails tends to unlock heterochromatin because it reduces the positive charge of histones. Since the DNA is charged negatively the connection between DNA and histones will become loose. The gene becomes switched on.

Overall the compacting or unlocking of genes is much more complicated involving both DNA methylation, histone tail modifications and the involvement of other proteins. Again the exact mechanism is not known at this time.

Non-coding RNAs are not epigenetic marks but they modify DNA transcription by binding to messenger DNA.

Especially interesting for us is the group of the micro RNA or miRNA for short(er). MiRNA do not alter the epigenetic state but they suppress or fine tune the gene expression. Short pieces of RNA are transcribed, transported out of the nucleus into the cytosol, and enzymatically shortened. Those short pieces of RNA then block the translation of mRNA into proteins and therefore influencing which protein is expressed or how much of it. The even more interesting part of miRNA is that they are packaged into vesicles and secreted into the bloodstream to communicate with other cells.

DNA methylation, histone tail modifications and non-coding RNAs work together to regulate DNA expressions. We do not know in what order or who is triggering what. At this point we only know that they trigger and modulate each other.

At this point you have a basic understanding how the epigenome works to regulate gene expression. Here is a summary:

1. Genes are suppressed or expressed by winding the corresponding piece of DNA around histones.

• Euchromatin: DNA wound loosely; gene accessible to enzymes. The gene is switched on.
• Heterochromatin: DNA wound tightly; gene inaccessible to enzymes. The genes is switched off.

2. Modulating how tight chromatin is packaged depends on the combination of epigenetic marks attached:

• Methyl groups attached to the DNA (tend to package tightly and turn genes off)
• Histone tail modification: Variety of chemical groups attached in various combinations to histone tails

3. The epigenome is regulated by:

• Epigenetic machinery (enzymes)
• Availability of epigenetic marks
• Non-coding RNA

The next step would be to look at how our diet can influence the epigenome.

The Diet Supplies Epigenetic Marks And Regulates the Epigenetic Machinery

Before we even talk about the dietary modification of the epigenome you need to understand the status of the current evidence: Epigenetic research is in the infant stages. It is an exciting field of research that will have major implications how eating and health needs to be understood. So far the evidence supports the big idea, while researchers only start to understand the complex detailed molecular mechanisms. It is very likely that the big idea will be tweaked and some aspects completely changed.

Nevertheless, the understanding of the relationship between diet and the epigenome already shifted how we are thinking about nutrition and is worth to have a look.

To understand how our diet modulates the epigenome, you need to separate the two components of the epigenetic regulation:

1. Pool of epigenetic marks (sometimes called tags) in the cell: The expression of the DNA is regulated by how tightly the double helix is wound around histones.  This is accomplished by attaching epigenetic tags either directly to the DNA (DNA methylation) or attaching  epigenetic tags to the histone tails (the histone modification). Every time a cell divides the tags need to be duplicated as well.
2. Epigenetic machinery: Epigenetic tags are attached by a set of enzymes. The amount of enzymes and the activity of enzymes can vary. Their expression and activity is fine-tuned by micro RNA. Together this is called the epigenetic machinery.

DNA methylation or histone modification can only take place if the cell has a sufficient pool of molecules that can serve as epigenetic marks. So far histone tail methylation and acetylation are the most researched. From this research we know that DNA  methylation tends to turn sections of DNA into heterochromatin, turning a gene off. Acetyl groups tend to turn genes on. Keep this in mind for the next part.

Let’s start with the pool of molecules that can be used as epigenetic marks:

Methyl groups come from dietary methyl donors. Two vitamins play a role here, folate and vitamin B12. For example research shows that newborns of women with folic acid supplementation during pregnancy have higher levels of DNA methylation. Other methyl donors are the amino acid methionine as well as betaine and choline.

The second group of tags are acetyl groups. Acetyl groups tend to come from the energy metabolism.  All surplus glucose and amino acids from the diet are fed into the pathways of the energy metabolism in the liver until they reach acetyl-CoA. Acetyl-CoA is then used for lipogenesis.

This means that over-nutrition tends to produce an acetyl-CoA in surplus in the cell. It is hypothesized that this acetyl-CoA surplus promotes an acetylation of histone tails and turns genes on. Starvation and excessive exercising on the other hand will reduce the availability of acetyl marks.

In addition to epigenetic tag availability, the diet can also influence the activity of the epigenetic enzyme machinery (remember that enzyme activity can be up- or down-regulated). This area is much less understood. Scientists think that metabolites from the citric acid cycle, phytochemicals and some nutrients regulate the epigenetic machinery. The most researched phytochemicals are the ones from tea, cruciferous plants and soy as well as resveratrol from blue and red fruits and vegetables. The field is rapidly expanding and will be exciting to watch.

The newest research area looking into the relationship between diet and epigenetic regulation of the genome is in this area of the gut microbiome. New research suggests that diet composition modulates the gut microbiome. If the diet supplies for example sufficient amounts of fiber the microbiota will produce ample amounts of short-chain-fatty-acids. Those SCFA modulate the epigenetic enzyme machinery. Scientists hypothesize that this is one explanation how diet and cancer are connected.

Just looking at this chart and thinking about the many ways the diet can vary between individuals it should become clear how complicated it is to research the connection between diet and epigenetic regulation of DNA expression. This level of complexity will require new approaches to research and definitively collaborations between several branches of science. Nutritionists and biochemists will not have the tools to research this level of complexity. It will require involvement of data scientists, programmer and biological systems engineers—just to name a few—to make progress.

Here is a summary:

Epigenetic regulation has two moving parts:

1. Epigenetic mark pool is determined by the diet

• • Methyl groups come from dietary methyl donors: Choline, betaine, folate, vitamin B12, some amino acids
  • Acetyl groups come from an energy surplus in the cell

2. Epigenetic machinery, a set of enzymes and miRNA, has the potential to be up- or down-regulated by

• • Phytochemicals
  • LCFA (long-chain fatty acids) from the gut microbiome (influenced by diet)

Sufficient availability of methyl groups is necessary to package genes tightly and switch them off.

Ample availability of energy is connected to the switching on of genes.

Is a Choline-Rich Diet the Key to Everlasting Life? Not so Fast, It’s More Complicated Than That

Epigenetics made it into the media and blogs during the last years. The main hypothesis journalists and bloggers latched onto is that a diet rich in choline (a major methyl donor) will promote methylation of DNA. Well-methylated DNA is supposed to suppress genes that code for genetic predisposition of chronic diseases such as T2D, CVD, and certain cancers.  These authors then suggest that eating a diet rich in animal products provides a lot of choline and is therefore beneficial for health. How much is factual scientific evidence?

Before even getting into the scientific facts. Do you know what foods provide methyl donors such as folate, vitamin B12, choline and betaine?

The table above lists methyl-donors and their food sources. A mixed, healthy diet will provide plenty of methyl donors. The problem is rather that folate and choline intake, both essential nutrients, is on the lower side in the US. The goal for everybody is a sufficient dietary intake of folate, B12, and choline from a healthy diet. Newer studies caution though that too much choline, predominantly from animal food sources, is metabolically connected to an increased risk for CVD.

Now to the evidence: Most of the research in this area was completed during pregnancy. The landmark study  showed that mice fed a diet with added choline during pregnancy resulted in pups that were less likely to be obese as adult mice.

That sounds great, but it’s not that easy. The mice used in this study are called Agouti mice. Agouti mice have a specific gene that makes them prone to obesity. The same gene also codes for the coat color.

If the gene is switched on the mouse is yellow and becomes obese very rapidly. If the gene is switched off the mouse is brown and lean. Adding choline to the mouses mother’s diet switches the gene off and results in thin brown mice offspring. This effect lasted through two generations of mice.

Obesity epidemic solved? Not even close. Keep in mind that this was a specific gene in those Agouti mice. The study demonstrated elegantly that genes can be switched on and off by feeding a methyl-rich diet. The study cannot prove that a methyl-rich diet will prevent obesity in humans.

After this landmark study many scientists started researching this hypothesis in animals, but also in pregnant women and confirmed that a sufficient supply of methyl groups during pregnancy results in a higher degree of methylated DNA in the fetus. The most advanced research in humans shows that folate supplementation is connected with higher DNA methylation in babies.

Other landmark observational studies looked at the connection between starvation or times of abundance and health outcomes in the next two generations. The results were much more inconsistent, but indicated that starvation and over-nutrition does have a negative impact on health for the next generations.

The current evidence supports the following big ideas:

• Supplying sufficient methyl groups during pregnancy—many American women have a diet low in methyl-donors—might be connected to better health for the next generations. These methyl groups can come from choline rich animal products, but studies also showed higher DNA methylation when folate (plant sources) was supplemented.
• Phytochemicals  from a plant-based diet and sufficient essential nutrients seem to be beneficial in maintaining health. This effect might reach far into the future of next generations.
• For most of those big ideas we are only at the beginning of understanding these mechanisms.
• Over-nutrition and starvation both increase the risk for chronic diseases in following generations. The evidence is more inconsistent.

What does this mean for eating decisions? The current evidence supports the recommendation to eat a healthy, mixed, plant-based diet and maintain a healthy weight.  Current evidence does not support a high meat (choline-rich) diet or even less genotype or phenotype specific dietary pattern as sometimes claimed in the media.

Epigenetic Changes Can Be Inherited to Children and Grandchildren During the Sensitive Periods

You know now that our diet has the potential to change epigenetic marks and influence the epigenetic enzyme machinery. This in turn, has the potential to change which genes are switched on and which genes are switched off. Lastly, there are studies that connect epigenetic changes to the risk for chronic diseases.

I also mentioned that it does not stop there. We saw in hunger studies that starvation can influence metabolic health of children and even grandchildren. Again, the research can give us a general idea, but not too many details at the moment.

Every time a cell divides most of the epigenetic marks are removed, but after cell division is completed those marks are re-established in the daughter cell in the same pattern as before. Nobody knows at the moment how, just that those marks are mitotically inherited from cell to cell.

This is important to know because it means that normally changes to the epigenome cannot be inherited to the next generation. Wait! Didn’t we just say that lifestyle decisions especially over- and under-nutrition can change chronic disease risk for children and grandchildren?

There are three exceptions. In order to understand how epigenome of our children and grandchildren can be altered by our lifestyle choices, we need to look at the epigenome during the early embryonal development.

There are two sensitive periods when the embryo is vulnerable.

First sensitive period: When the sperm meets an oocyte the zygote forms and starts dividing. Those cells need to develop into all sorts of tissues and need to be totipotent. This is achieved by removing almost all of the epigenetic marks resulting in the deletion of most of the epigenome.

The embryo keeps developing and different cells are assigned to different tissues. In the process the epigenetic marks are reset to establish an epigenome for different tissue types as inherited by the parents. Nobody has an idea how the cells knows how.

This time of re-establishing the epigenome according to the blueprint is called a sensitive period. External influences during this short window in the life of an embryo such as over- or undernutrition of the mother or a diet low in methyl donors, can change the resetting of the epigenome. Once the epigenome is established it will be duplicated during each cell division. On a side note, random failure to establish correct epigenetic marks will lead to the loss of the embryo, some childhood cancers, or epigenetic diseases.

The second sensitive period also takes place during fetal development. This time,  future grandchildren are affected.

During embryonal and fetal development the reproductive system is created. As part of this development, cells grow into primordial germ cells that will later develop into oocytes and sperm cell. Cells destined to be germ cells need to go from a diploid cell to a haploid cell. During this process most of the epigenetic tags are removed and then re-established in the germ cell. This second sensitive period takes place during mid gestation and is also sensitive to epigenetic mark changes. Lifestyle of the pregnant mother will now influence the metabolic health of a potential future grandchild.

The third sensitive period happens only in boys. Girls are born with developed oocytes and undergo the entire germ cell development in utero. In boys the germ cell goes dormant until the boy  starts producing sperm during puberty. Early puberty in boys is the last time when epigenetic marks are mostly deleted. The boy’s lifestyle—availability of methyl donors, over-or undernutrition— might influence the resetting of epigenetic marks and therefore the health of his future child.

The Epigenome Changes Profoundly Throughout Life

Remember that I stated before that during cell division the epigenome is duplicated so the daughter and mother cell are identical. This is generally true, but not completely.

Based on the evidence we have today we can hypothesize that the epigenome responds to our lifestyle and the environment we are living in. This seems to be the purpose of the epigenome.

From studies looking at the epigenome throughout life in lab animals we know that the epigenome changes profoundly. We also see changes in humans and this explains why identical twins start out fairly identical but then start becoming their own epigenetic person. They will look slightly different and develop different disease risk for example.

These continuous changes are not inheritable to the next generation.

Epigenetic changes throughout life can happen two ways:

• Unintentionally: During cell division errors are made when epigenetic marks are copied from the mother to the daughter cell. If the error is grave, apoptosis might be induced, but smaller mistakes are now duplicated when the daughter cell starts dividing. We think that this might be one way that cancer starts. Errors transferring epigenetic marks that suppress a gene coding for a cancer predisposition might accidentally unlock this gene. Those errors accumulate with age and it is thought that this is one of the reasons for an increased disease risk during aging.
• Intentionally:  The hypothesis is that organisms need to detect environmental changes and adapt if they want to survive. For example lack of food for a prolonged time might trigger changes making the  metabolism more energy efficient.

In summary:

• There are 3 sensitive periods when the epigenome can be modified during the re-establishing of the epigenetic marks:

1. 1. Fertilization to ~6 weeks when most of the epigenome is removed to allow totipotency.
   2. Week 9-16 when the primordial germ cells are established in the embryo. The epigenetic marks are again removed from the cells intended to become germ cells.
   3. In boys the beginning of puberty when sperm production starts.

Major events like starvation, over-nutrition or nutrient deficits can influence the epigenome. Changes to the epigenetic marks in germ cells or the embryo during these sensitive periods will be replicated during cell division and therefore change the phenotype.

• Epigenetic marks change over our lifetime due to copy errors while cells are dividing and the availability of molecules that can be used as epigenetic tags.

Research in this area is exciting and will enable us to explain many hypotheses regarding chronic diseases we were not able to explain so far:

Interested In More Information?

• Mitosis: https://youtu.be/C6hn3sA0ip0: https://youtu.be/C6hn3sA0ip0
• Transcription: https://youtu.be/SMtWvDbfHLo