Epigenetics: DNA Methylation and Histone Modification

Epigenetics is the study of heritable changes in gene activity that do not involve changes in the DNA sequence, and its main mechanisms are DNA methylation, which usually silences genes, and histone modification, which changes how tightly DNA is packed. In brief: every cell of an organism has the same DNA, but different cells switch different genes on and off, and epigenetic marks help to keep these patterns stable when cells divide. Marks can be affected by diet, stress, smoking and other environmental factors, which is one way that genes and environment interact. Some marks are reset in the germline, but a few may be passed to offspring.

Epigenetics helps to explain how identical twins diverge, how cells keep their identity and why some diseases such as cancer involve gene regulation. This guide reviews gene regulation, explains DNA methylation and histone modifications, covers X-inactivation and genomic imprinting, discusses gene-environment interactions, works through an interpretation example and corrects common mistakes.

How are genes regulated at the level of chromatin?

In the nucleus, DNA is wrapped around histone proteins to form nucleosomes (about 147 base pairs of DNA around a core of eight histones), and the nucleosomes are packed into chromatin. Chromatin exists in two broad forms:

• Euchromatin: loosely packed, accessible to transcription factors and RNA polymerase, and typically active.
• Heterochromatin: tightly packed, inaccessible, and typically silent. Centromeres and telomeres are heterochromatic.

Epigenetic marks are chemical tags that change the packing and the accessibility of genes, without changing the base sequence. The word comes from the Greek epi, “above” or “on top of”, genetics. The marks are added by “writer” enzymes, recognized by “reader” proteins and removed by “eraser” enzymes, which allows them to be dynamic.

What is DNA methylation?

In DNA methylation, enzymes called DNA methyltransferases (DNMTs) add a methyl group (–CH₃) to the base cytosine, usually where a cytosine is followed by a guanine (a CpG site). In humans, about 70 to 80 percent of CpG sites are methylated. Stretches with many CpG sites, called CpG islands, are found near the start of about 60 percent of human genes.

• Methylation of a CpG island in the promoter generally silences the gene: it blocks the binding of transcription factors, and attracts proteins that bind methylated DNA and recruit enzymes that make chromatin compact.
• Unmethylated promoters are usually active.
• Maintenance: after DNA replication, the new strand is unmethylated at first. The enzyme DNMT1 recognizes the hemimethylated sites and copies the pattern onto the new strand, which allows the pattern to be passed on to daughter cells. This is the basis of cellular memory.
• Demethylation: TET enzymes remove methyl groups, which allows genes to be reactivated.

What is histone modification?

The tails of histone proteins stick out of the nucleosome and can be chemically modified at specific amino acids, mainly lysine. The main modifications are:

• Acetylation (adding an acetyl group, by histone acetyltransferases, HATs): removes the positive charge of lysine, which weakens the attraction between the histones and the negatively charged DNA. Chromatin loosens, and genes are generally switched on. Histone deacetylases (HDACs) remove the acetyl groups and silence genes.
• Methylation of histones: its effect depends on the site and number of methyl groups. For example, trimethylation of lysine 4 on histone H3 (H3K4me3) marks active promoters, whereas trimethylation of lysine 9 or 27 (H3K9me3, H3K27me3) is associated with silencing.
• Phosphorylation and ubiquitination: involved in cell division and DNA repair.

The combination of marks is sometimes called the “histone code”. DNA methylation and histone modifications work together: for example, methylated DNA recruits HDACs, which remove acetyl groups, making the chromatin compact. Non-coding RNAs, such as the long RNA Xist, are also involved in epigenetic regulation.

Epigenetic mechanisms compared

Where do epigenetic mechanisms matter?

• Cell differentiation: as a stem cell becomes a specialized cell, genes that are not needed are silenced by epigenetic marks. Reprogramming to iPSCs removes many of these marks.
• X-chromosome inactivation: in female mammals, one X chromosome in each cell is silenced early in development by the RNA Xist and heavy methylation, forming a compact Barr body. As the choice is random in each cell, a female is a mosaic, as in tortoiseshell cats.
• Genomic imprinting: for a few hundred genes, only one of the two copies, either the maternal or the paternal copy, is active, because the other is silenced by methylation in the germline. Errors cause disorders: Prader-Willi syndrome and Angelman syndrome are associated with a deletion in the same region of chromosome 15, which causes different syndromes depending on whether it is inherited from the father or the mother.
• Cancer: tumors often show global loss of methylation (which can activate oncogenes and cause genome instability), together with hypermethylation of tumor-suppressor gene promoters (which silences them). Drugs that inhibit DNMTs or HDACs, such as azacitidine, are used in some blood cancers. Methylation patterns in blood DNA are being developed as biomarkers for early cancer detection.
• Ageing: methylation changes with age in a predictable way, which forms the basis of “epigenetic clocks” that estimate biological age.

How do gene-environment interactions work?

Epigenetic marks respond to the environment, and so link experience with gene activity. Examples:

• Agouti mice: in a classic experiment by Robert Waterland and Randy Jirtle (2003), pregnant mice fed a diet rich in methyl donors (folic acid, vitamin B12, choline) produced offspring with more methylation of the agouti gene, and brown, healthy coats, instead of yellow, obese coats.
• Dutch Hunger Winter (1944 to 1945): people who were conceived or in early gestation during the famine had, decades later, differences in the methylation of certain genes and higher rates of obesity and heart disease. The cause-and-effect relationships are still being studied.
• Twins: identical twins are epigenetically more similar when young. They diverge with age, and differences are greater in twins who live apart and have different lifestyles (a study of Mario Fraga and colleagues in 2005).
• Maternal care in rats: pups that were licked and groomed more by their mothers had a different methylation of a stress-hormone receptor gene, and were calmer adults (Michael Meaney and colleagues).

Transgenerational epigenetic inheritance, which means marks that are passed through the germline to the next generation, is well documented in plants and in some animals such as worms. In mammals, most marks are erased in the germ cells and early embryo, and evidence of such inheritance in humans is limited and debated. Claims that experiences of grandparents change the health of grandchildren must be treated with caution.

Worked example: interpreting methylation data

A researcher compares the promoter of a tumor-suppressor gene in normal and tumor cells. For each sample, 20 CpG sites in the promoter are tested, and the number found to be methylated is counted. Expression of the gene is measured by the amount of mRNA.

1. Percentages: 2 ÷ 20 = 10 percent; 15 ÷ 20 = 75 percent; 18 ÷ 20 = 90 percent; 3 ÷ 20 = 15 percent.
2. Pattern: as methylation rises from 10 to 75 and 90 percent, the mRNA falls from 100 to 12 and 4 units. This negative relationship is consistent with silencing of the gene by promoter methylation.
3. Tumor 3: low methylation (15 percent) and nearly normal expression, so in this tumor the gene is not silenced by methylation. Another mechanism, such as a mutation in the gene, might disable it, or the tumor might depend on other genes.
4. Conclusion and caution: the data show a correlation in three tumors, not proof of causation. A test of cause would be to treat the cells with a demethylating drug and to see whether the expression of the gene is restored.

The same logic applies in the use of methylation as a biomarker: a pattern in blood or tissue is compared with a threshold. A small number of samples, as here, would not be enough for a firm conclusion.

How did the field develop?

Conrad Waddington coined the word epigenetics in the 1940s to describe how genes interact with the environment to produce phenotypes, with his image of a ball rolling down an “epigenetic landscape” of valleys. Mary Lyon proposed the random inactivation of one X chromosome in female mammals in 1961. Robin Holliday and Arthur Riggs suggested in 1975 that DNA methylation could be a heritable mechanism of gene control. The Human Epigenome Project and projects such as ENCODE and Roadmap Epigenomics mapped marks in the 2000s and 2010s.

Which mistakes are common?

Mistake 1: Saying that epigenetic changes alter the DNA sequence

They do not. They change chemical tags on DNA or histones.

Mistake 2: Believing that methylation always silences genes

Methylation of promoters generally silences, but methylation within genes has other effects, and histone methylation can activate or silence.

Mistake 3: Thinking that epigenetic marks are permanent and always inherited

Many are reversible, and most are reset between generations in mammals.

Mistake 4: Treating epigenetics as a replacement for genetics

It adds a layer of regulation. The DNA sequence remains the basic information.