Epigenetics has become something of a buzzword in recent years. The unknowns far outweigh the knowns in the young field of epigenetics, which is part of what makes it such an exciting time and fascinating subject.

Speaking about numbers, epigenetics publications have reached 5,000 papers in 2018, when they were inexistant at the beginning of our new millenium.

But what is Epigenetics ?

"Epi" is Greek for "above," therefore, meaning modifications above the genes or on the genes. In 1957, epigenetics was first introduced by Conrad Waddington, a biologist who studied how similar genotypes affected a wide variety of phenotypes.

Epigenetics is the study of changes in organisms caused by modification of gene expression, rather than an alteration of the genetic code itself. It is a dynamic method that allows an organism to react phenotypically to its environment by expressing or silencing certain genes in response to its environment.

Epigenetics forms a layer of control that determines which genes are turned off and which genes are turned on in particular cells in the body. They do this by making chemical modifications of chromosomal DNA and/or structures that change the pattern of gene expression without altering the DNA sequence.

Epigenetics in action :

from caterpillar to butterfly, the same gene set for a totally different bug

But epigenetics still present many challenges and mysteries :

• The lack of omnipresence : whereas a blood sample can be used to 'genotype' an individual, most epigenetic marks in blood DNA provide no clues about epigenetic dysregulation in other parts of the body, such as the brain or heart, 

• A massive amount of data to treat : new atlas to map DNA methylation for instance required 370 times more data than were used for the first map of the human genome in 2001,

• Unclear inheritance mechanims :

In mammals, epigenetic marks are erased completely, and reprogrammed twice during the lifetime of an individual. The first wave of epigenetic erasure happens in primordial germ cells. Then the methylation comes back again in egg-specific marks and sperm-specific marks. And then, upon fertilization, that egg and that sperm meet and the marks are erased again. So there’s finally two rounds of epigenetic reprogramming that occur in the germline that basically prevent any epigenetic marks from being transmitted from one generation to the next.

Garvan Institute of Medical Research recently suggests that a particular epigenetic change, that deactivates some genes linked to cancer late in human development (control of CTA genes) has been conserved for more than 400 million years, passing from zebra fish to humans throughout evolution.

But how does epigenome is copied when DNA gets copied ? Very difficult to say.

Regarding the trends of epigenetics field, we can note different paths which should be noted :

• novel epigenetic mechanisms of gene regulation,
• new approaches and methods to investigate epigenetics.

The main epigenetic mechanisms studied in mammalian cells are DNA methylation and histones modifications which induce remodeling of chromatin resulting in changes of cellular phenotypes.

While post-translational modifications of histones could probably be considered the “rock stars” of epigenetics, the key’s really how those modifications change the structural properties of chromatin giving access to DNA regulation factors.

Although we counted over 170 epigenetic modifications to RNA, N6-methyladenosine (m6A) is the most abundant and has therefore been in the spotlight the last few years. The biological roles of m6A are still being clarified, but it appears to be enriched in untranslated regions (UTRs) of the transcriptome and its levels and localization change during several cellular processes such as stem cell differentiation, tumorigenesis, and the stress response.

Long non-coding RNAs (lncRNAs) are RNA molecules that are over 200 nucleotides long and which do not encode proteins. These molecules regulate gene expression and, in humans, are over four times more abundant than coding RNA sequences. For the moment they’re still poorly understood epigenetic molecules, but will be more and more studied in the future.

Continuing with the RNA modification theme, another epigenetic modification of RNA, N4-acetylcytidine (ac4C), has been identified as a new modification to mRNA catalyzed by the enzyme N acetyltransferase 10 (NAT10). The ac4C modification is mainly found on transfer RNA and ribosomal RNA molecules, but recent reports have hinted at this modification also being present in mRNAs.

Two studies published in December 2018 revealed that the H3K4/H3K36 methyltransferase SETD3 is responsible for methylation of β-actin H73. Methylation of histidine has previously been observed in several proteins, but its function remains enigmatic.

ATAC-seq means Assay for Transposase-Accessible Chromatin using SEQuencing and was first described in 2013. ATAC-seq makes use of a hyperactive mutant Tn5 transposase that can simultaneously fragment and tag DNA in open chromatin with a sequencing adapter. The advantage over MNase or DNase-Seq lies simultaneously in the small amount of cells required and the speed of the test.

Bio-Rad has released this summer its Single-Cell ATAC-Seq machine building on the potential of microfluidic technology, but we can also combine multiplexed CRISPR technology with genome-wide chromatin accessibility profiling in single cells, thanks to SC-Perturb-ATAC-Seq method.

Mass cytometry is a single-cell proteomic technology that allows for the analysis of 40+ cellular parameters using antibodies tagged with heavy metal isotopes. CyTOF® technology overcomes the limitations of fluorescence-based detection modalities by separating signals based on differences in mass instead of wavelength. CyTOF offers many advantages, including :

• Extremely low detection limits
• A large linear range
• Possibilities to detect isotope composition of elements
• High sample throughput – speed

EpiTOF is the application of mass cytometry to epigenetics, a multiplexed mass cytometry assay that can investigate the global levels of 40 different histone modifications in T cell samples at single-cell resolution.

ChIP-seq is a useful technique that allows researchers to interrogate the physical binding interactions between protein and DNA using next-generation sequencing. ChIP makes use of reversible cross-links made between DNA and associated proteins. The fixed chromatin is physically sheared and DNA fragments associated with a particular protein are selectively immunoprecipitated and analysed.

ChIP-Seq offers many adavantages :

• Captures DNA targets for transcription factors or histone modifications across the entire genome of any organism
• Target transcription factor binding sites
• Reveals gene regulatory networks in combination with RNA sequencing and methylation analysis
• Offers compatibility with various input DNA samples

But ChIP-Seq does have some limitations. Most notably, it is often difficult to immunoprecipitate enough material from low cell numbers to generate high-quality next-generation sequencing data.

A group of researchers in Japan rose to the challenge and developed a new method called “ChIL”, which stands for Chromatin Integration Labelling. Rather than performing a chromatin preparation followed by an IP reaction, ChIL instead uses a primary antibody for the target of interest followed by addition of the ChIL probe. A ChIL probe is a secondary antibody that recognizes the first antibody conjugated to DNA sequences required for amplification and library preparation.

CONCLUSION

Some of the most important areas of epigenetic innovation are new approaches for single-cell analyses and higher throughput genomic and epigenomic assays with increasingly higher resolution.

Epigenetics research is an extremely dynamic field and more and more studies are published including recent papers on :

• Nurture Vs Nature
• Rescue for Cell-Based CAR T Therapy
• Aging process thanks to « Epigenetic clocks »
• Drug addiction
• Exposure to weapons of mass destruction
• Social Epigenetics
• Allergy and epigenetics.

Following the completion of the Human Genome Project, the Human Epigenome Project is currently striving to map the scope of changes that can occur between genome and phenotype.