ABOVE: A model of the nucleosome

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John D. Loike, a Professor of Biology at Touro College and University Systems, writes a regular column on bioethics for The Scientist.

Epigenetics, the study of mechanisms by which genes are turned on or off without altering their genetic code or DNA sequences, is one of many ways that cells regulate gene expression. Epigenetics has helped scientists better understand complex and diverse biological processes such as cell differentiation, genomic imprinting, and X-chromosome inactivation and operates via two mechanistic processes: a) histone modifications (for example, methylation, acetylation, ubiquitination, and phosphorylation) and b) direct methylation of cytosine base pairs.

Two new methods of epigenetic assessment and intervention, APOBEC-coupled epigenetic sequencing (ACE-seq) and CRISPR, have the potential to dramatically enhance epigenetic research and its clinical applications.

Described in Nature Biotechnology last month, ACE-seq is a bisulfite-free method for localizing 5-hydroxymethylcytosine at single-base resolution with low DNA input and without harming DNA. Until recently, bisulfite treatment of DNA followed by PCR amplification was the gold-standard epigenetic assay to detect methylated cytosines, but in doing so at extreme pH and elevated temperature, the protocol can destroy the sample. ACE-seq, on the other hand, uses one member of the AID/APOBEC family of enzymes that catalyzes the deamination of cytosine to uracil in single-stranded DNA and has high activity and a particular proficiency for 5-hydroxymethylcytosine deamination. Upon sequencing the sample after the enzyme treatment, users can discriminate 5-hydroxymethylcytosine from cytosine and 5-methylcytosine by scanning for uracils.

On a global level these methods may help us better establish whether a specific epigenetic mark causes a change in gene expression or simply correlates with changes in genetic activity.

ACE-seq offers several advantages over bisulfite treatment and can achieve the same effect as bisulfite without degrading the sample. ACE-seq is better at identifying common and uncommon epigenetic modifications and facilitates sequencing the entire genome.

In 2017, scientists from the Salk Institute for Biological Studies in California reported a robust CRISPR-Cas9–based system for activating target genes in vivo by modulating histone modifications rather than by editing DNA sequences. They found that their system was successful in ameliorating disease symptoms in mouse models of diabetes and muscular dystrophy. Prior to this paper, most approaches to alter epigenetic processes relied on drugs that ubiquitously add or remove histone modifications, potentially affecting off-target genes and producing serious side effects. Other groups also are using modified CRISPR-Cas9 complexes to rewrite histone marks by inducing methylation or acetylation at nucleosome level.

Using ACE-seq and CRISPR-Cas9 technologies, scientists will have better tools to explore fundamental questions related to epigenetics. On a global level these methods may help us better establish whether a specific epigenetic mark causes a change in gene expression or simply correlates with changes in genetic activity, and which epigenetic marks act synergistically with one another or epistatically. More specifically, these techniques can address many unresolved questions. First, under what conditions do epigenetic changes affect other mechanisms of gene regulation, such as transcription factors, gene-gene interactions, and noncoding RNAs that can block transcription? Second, under what conditions are epigenetic changes transmitted to offspring? Third, what are the precise mechanisms that specific environmental factors target one or several genes to generate an altered phenotypic outcome? Fourth, what determines the cell type or tissue targeted by environmentally triggered epigenetics? And finally, under what conditions can epigenetic patterns be reversed by changes in lifestyle or drugs? Examining all of these questions will enable physicians to better understand how to incorporate epigenetic information into medical diagnosis and therapy options.

The utilization of technologies that explore epigenetics already has many immediate applications in medicine, including: a) cancer chemotherapy, b) analyzing the health of embryos obtained naturally versus via IVF, c) tumor diagnosis, and d) the onset of neurological diseases such as Alzheimer’s disease. The range of diseases that may be treatable by epigenetic protocols may explain why companies, such as Gotham Therapeutics, have launched multi-million-dollar drug discovery programs that focus on discovering epigenetic-modulating drugs. Several companies (for instance, Epigenomics) and research centers assess epigenetic signatures in cell-free DNA obtained from the blood of patients with cancer to identify the tissue origin of the tumor.

If epigenetic research utilizing these new technologies will successfully shed some light in disease prevention, diagnosis, and therapy, then the research can expand to study epigenetics related to human behavior and moods. Aggression, violence, adultery, sexual preferences, risk-taking, happiness, depression, and even spirituality may all be affected by gene regulation, including epigenetics, via mechanisms not yet precisely defined. There also is much evidence that diet, sleep, fasting, exercise, and stress regulate gene expression but here, too, the way they do it needs to be explored.

Incorporating these new epigenetic technologies when examining the multiple biological factors that regulate gene expression will better illuminate whether or how environmental factors and lifestyles can modify what we classically believed was our DNA destiny.