Demethylation in Ageing and Disease

When it comes to genetics and nutrigenomics the focus has predominantly been on methylation. Methylation is the process by which a methyl-group is added to a molecule such as an amino acid or a vitamin.

But what about demethylation? What is demethylation and what is the difference between the two?

To simplify…

Methylation is the ‘switching off’ of genes.
Demethylation is more like the ‘clean up crew’ that sanitizes DNA methylation imprints for the next generation.

The collection of all your genes and DNA is called your genome. Your genome contains all the information needed to build and maintain your cells and your body. You carry a copy of your entire genome, which consists of more than 3 billion DNA base pairs, inside every one of your cells that have a nucleus.

Your genome needs to be both stable (to protect you and future generations) and flexible (to accommodate for change).

DNA METHYLATION

According to Wikipedia:

“DNA methylation is a process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription.”

DNA methylation is incredibly important for cell division and keeping our genes stable.

We need cell division for:

Fertility
Growth
Wound healing and repair
Repairing organs
Healing of a compromised gut barrier (leaky gut)
Growth of hair and nails

We need to keep our genes stable to prevent or slow down:

Chronic disease
Ageing
Cancer

All of this is regulated by this biochemical process called DNA methylation. A very important process as we can see.

DNA methylation in early embryonic development is regulated by two enzymes namely DNMT3a and DNMT3b, and maintained by the enzyme DNMT1. All of these are DNA Methyltransferase enzymes (DNMT’s) that require SAMe (S-adenosylmethionine) to function like all other methyltransferase enzymes. SAMe is a by-product produced from the metabolic methylation cycle.

DNA is made up of various combinations of four nucleotides, namely adenine, guanine, cytosine and thymine. You may have seen images of DNA before where it looks like the steps of a ladder that are twisted around each other. The four nucleotides mentioned make up these steps.

DNMT enzymes have both the ability to introduce methylation marks on our DNA as well as maintain methylation after the genome is replicated. As an example, DNMT enzymes are responsible for converting the nucleotide cytosine (C) into 5-methylcytosine (5mC) by attaching a methyl-group to position-5 of the cytosine DNA base.

5-Methylcytosine (5mC) is often used as a marker to protect our DNA from being cut by methylation-sensitive restriction enzymes. When cytosine is methylated, our DNA will maintain the same sequence or format even though the expression can still be altered or changed. This is important as it provides genome stability.

DNA DEMETHYLATION

DNA Demethylation is the reversal of this process as the name implies, or a removal of a methyl-group from cytosine nucleotides.

DNA Demethylation can be both passive or active.

Passive demethylation occurs when 5-methylcytosine (5mC) becomes lost during cellular replication. There is a high demand for methylation and SAMe production during cellular replication as DNMT needs a lot of SAMe to methylate newly formed DNA. If the need for repair outnumbers our cell’s ability to keep up with methylation and SAMe production, DNA methylation will become dysregulated or non-functional. The consequence is that the genome loses its stability and becomes unstable. Once unstable it becomes easier to retain damaged or faulty DNA within the genome which then gets replicated leading to a cohort of cells with faulty DNA. This eventually leads to the development of chronic disease and even cancer.

Passive DNA demethylation occurs when you run out of SAMe.

Active demethylation involves enzymes that either removes or modifies the methyl-group on 5-methylcytosine (5mC) for a purpose. Let’s look at this process further…

TET ENZYMES AND EPIGENETICS

The discovery of TET (Ten-eleven Translocation) enzymes has been one of the most important discoveries in epigenetics. It showed that there is in fact such a process as active DNA Demethylation and also how it occurs via oxidation.

Interestingly translocation in cancers commonly occur between chromosomes 10 and 11 which is where these TET enzymes got their name from. Ten-eleven Translocation enzymes.

Essentially TET enzymes have the ability to remove methyl-groups from our DNA, more specifically the cytosine nucleotide of DNA, which makes cytosine modification very important in the process of DNA methylation and repair.

It specifically converts 5-methylcytosine or 5mC (which is methylated cytosine as the name suggests) into 5-hydroxymethylcytosine or 5hmC (adding a hydroxyl group to it), then 5-formylcytosine (5fC), and finally into 5-carboxylcytosine (5caC). In general you don’t see much of these last two products, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) inside the cells unless TET enzyme function becomes overexpressed. The focus of research seems to be more on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).

As mentioned before TET enzymes are oxidation enzymes which means they use oxygen inside the cells to do their job. Oxygen is important! But they also need vitamin C as a co-factor. This could be one of the reasons why vitamin C is often used in cancer treatments as we’ll see later.

TET enzymes are commonly found in embryonic stem cells where they play a role in early embryonic development where its function is to demethylate the father’s chromosomes.

WHY ACTIVE DEMETHYLATION IS IMPORTANT

Active Demethylation Process #1

When sperm penetrates the egg the father’s DNA undergoes heavy remodelling and loses a lot of its 5-methylcytosine (5mC) in the process. Only once the remodelling is done does it merge with the mother’s DNA to form the baby’s new unique DNA foot print. The mother’s DNA remains unchanged throughout this process.

Active Demethylation Process #2

The fertilized egg becomes implanted in the uterine wall of the mother. During early development of the fertilized egg primordial germ cells (PGC’s) have to be reprogrammed so they can participate in meiosis. Meiosis is cell division that occurs during embryonic development where four daughter cells are formed each containing half the number of chromosomes of the parent cell. During this reprogramming process all the DNA methylation patterns are wiped from the cells, similar to wiping the memory of a computer.

This process of wiping the memory slate clean in a cell’s DNA is also called pluripotency.

Active Demethylation Process #3

Active demethylation can also occur when cells are exposed to intense environmental stimuli such as extremes in temperature, although a lot of the studies were done on plants.

All of these active demethylation processes will convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). So, it makes sense that there will be lower levels of 5-methylcytosine (5mC) and higher levels of 5-hydroxymethylcytosine (5hmC) found during the developmental process of the baby whilst in the mother’s uterus. 5-Hydroxymethylcytosine levels need to be high during these stages and when levels drop too low it will impair self-renewal in embryonic stem cells.

BRAIN

The second product produced by TET enzymes, 5-hydroxymethylcytosine (5hmC), is found in abundance in the human brain which is now being studied for its regulation of gene expression inside the brain. it represents an epigenetic mark by itself that may regulate chromatin structure and transcription. There is approximately 40% more 5-hydroxymethylcytosine (5hmC) in the neuronal Purkinje cells than 5-methylcytosine (5mC).

What does this mean?

Purkinje cells are a class of GABAergic neurons located in the cerebellum and are some of the largest neurons in the human brain. They are aligned like a stack of dominos and are largely responsible for motor coordination in the cerebellar cortex. They have been found damaged in:

Autoimmune disease
Spinocerebellar ataxia
Gluten ataxia
Autism
Alzheimer’s disease
Other neurodegenerative diseases

It raises the interesting question on the role of DNA methylation and demethylation when it comes to neurodegenerative diseases and even autism as well as anxiety and sleeping disorders, since these are also regulated by the neurotransmitter GABA.

5-Hydroxymethylcytosine (5hmC) will become depleted during active DNA replication. This is because many of these cytosine derivatives such as 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) will be converted back to the nucleotide cytosine again through various processes in the formation of new DNA in new cells. This may have detrimental affects on cells such as the brain cells where 5-hydroxymethylcytosine (5hmC) has an epigenetic role in regulating gene expression in the brain.

This may occur during:

Growth
Cancer
Cell repair – leaky gut, wounds, damaged organs or cells

It is important that you support your body and biochemistry with the appropriate nutrition during these occurrences.

Growing children need good nutrition and not the junk food diets that they tend to grow up with in this modern age.
Those battling cancer, are in remission, or are at high risk of getting cancer need to make sure they get the nutrients in for both methylation and demethylation, and not just focus on chemotherapy and radiation.
If you are struggling to heal your gut, or wounds are slow to heal, then consider that you may be lacking in some of the nutrients needed for the processes we talked about.

WHEN SAMe (S-adenosylmethionine) IS NOT AVAILABLE

Remember we mentioned that DNMT enzymes use SAMe to methylate DNA. What happens when SAMe is not available or in short supply such as during nutritional deficiencies?

The current theory is that it is possible that DNMT may be involved in the demethylation of DNA instead of methylation if it doesn’t have its co-factor SAMe available. DNMT’s can potentially react with 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) and demethylate them further. If this in fact is the case it would make DNMT enzymes bi-functional where they can contribute to both functions, and thus very important when it comes to DNA expression.

CANCER

DNA methylation dysregulation sets the stage for cancer development. Dysregulation can come from:

Nutrient deficiencies – poor diet, poor gut absorption
Environmental toxins – metals, amalgam teeth fillings, lead paints, glyphosates, parabens in cosmetics, the 200,000 chemicals that get registered every week without being tested for safety
Infections – viruses, bacteria, parasites, fungi
Stress – overtraining, unhealthy relationships, financial, job, trauma, PTSD (post traumatic stress disorder)

Both 5-Hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) can be used as markers to measure cancer progression.

The less 5-hydroxymethylcytosine (5hmC) and the more 5-methylcytosine (5mC) you have, the more aggressive cancer becomes and the faster it grows.

This means we may prefer TET enzymes to be upregulated during cancer as it will convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). TET enzymes require vitamin C as a co-factor to work which is why there are so many favourable studies looking at vitamin C and cancer.

A recent paper published in Clinical Cancer Research, March 2018, Volume 24, Issue 6 titled Epigenetic Reprogramming Strategies to Reverse Global Loss of 5-Hydroxymethylcytosine, a Prognostic Factor for Poor Survival in High-grade Serous Ovarian Cancer, stated:

“New studies suggest that global 5-hmC levels, rather than individual genes and promoter methylation, show a better correlation with clinical outcomes in tumors.”

This is a very important statement because many women with the BRCA gene implicated in breast and ovarian cancer live in fear of one day contracting one of these deadly cancers. This statement essentially means that maybe we shouldn’t focus on the individual gene like it is a death sentence, but rather monitor other markers such as 5-hydroxymethylcytosine (5hmC) which can give us more information on when gene expression occurs and the progress of disease and/or treatment.

THE HYPO- AND HYPERMETHYLATION CONUNDRUM IN CANCER

There has been a lot of debate and confusion around methylation and cancer. Many cancer drugs such as tamoxifen are methylation inhibitors since methylation becomes heavily upregulated inside tumor cells to fuel growth of the cancercerous tumor. Some even go as far as to say that those with cancer should avoid folic acid supplements.

First of all, folic acid is synthetic and not good for anyone to take. We should rather opt for food-based folate or methylated folate supplements such as folinic acid and 5-methyltetrahydrofolate. But that is a discussion for another day.

What is important to understand is that global hypomethylation is the first step towards cancer development. Remember that methylation is needed to switch genes ‘off’ which includes cancer genes. If your methylation cycle is slowed or functioning poorly because you are not eating nutritious foods, not absorbing your nutrients, have infections you can’t get rid of, or are exposed to chemicals on a continual bases, then you are at an increased risk of developing cancer.

ROS (reactive oxygen species) or toxins react and damage the epigenetic mark, 5-methylcytosine.

Once cancer cells are formed and start to grow, they will rob your cells further of nutrients so they can speed up their own methylation cycle in order to grow faster. The tumor cells essentially become hypermethylated.

This is why we often see both hyper- and hypomethylation in the context of cancer.

This makes DNMT a very interesting enzyme, because we see how important DNMT is to methylate DNA and switch ‘off’ cancer causing genes and how it needs SAMe to do this job. When SAMe is in short supply, methylation inside normal cells slow down further diminishing SAMe supplies, DNA become unstable resulting in damaged DNA being replicated and the increased risk of cancer, and DNMT switches its function as a methylating enzyme to a demethylating enzyme.

SUMMARY

Both metabolic methylation and DNA methylation are important in keeping us healthy, to help us detoxify and to switch disease genes ‘off’. We require specific nutrients for these processes which include:

Folate – green leafy vegetables, eggs
B vitamins – beef, brewers yeast, lamb, legumes, liver, nuts, spirulina
Zinc – beef, bilberry, brewer’s yeast, egg yolks, liver, lamb, oysters, sunflower and pumpkin seeds, sea food
Magnesium – almonds, barley, brewer’s yeast, cocoa, cod, lima beans, parsnips, kelp, eggs, seeds
Amino acids (cysteine, methionine, serine, glycine) – protein, red meat, chicken, fish, pork

DNA Demethylation is another very important mechanism needed during embryonic development and to prevent cancer progression, as well as wiping epigenetic memory from our cells so that they can be reprogrammed. The TET enzymes responsible for demethylation requires specific substrates and cofactors to work, such as:

Oxygen – deep breathing, exercise
Iron (co-factor for TET proteins)
Vitamin C (enhances TET activity as a co-factor) – broccoli, brussel sprouts, citrus fruit, guava, parsley, rosehips, cabbage, sweet potatoes, blackcurrant
Vitamin A (activates TET2 and TET3 expression, thus increasing 5-hydroxymethylcytosine production) – barley grass, butter, carrots, cod liver oil, green leafy vegetables, liver, egg yolk

The take home message is to make sure you eat a nutritious diet and get plenty of these nutrients in order to support your body in regulating these processes.