Epigenetics

Epigenetics – A Hot Topic in Scientific Research

If you follow scientific literature in peer-reviewed journals, as well as mainstream news and social media, you have certainly heard of epigenetics. Epigenetics has been linked to human conditions such as cancer, obesity, diabetes and depression, and is an active area for drug discovery.

What is epigenetics?

In the late 1940s, Conrad Waddington introduced the term “epigenetic” (derived from “epigenesis”) as a branch of biology for the study of the interaction between genes and their products, related to the phenotype. Currently, epigenetics refers to chemical modifications of DNA and the associated histone proteins that control gene activity, independent of the genetic code. On a molecular level, there are dozens of identified proteins from animals and plants which are involved in creating, recognizing, and removing these epigenetic modifications (also called “marks”) on the histones and DNA making up chromatin.  These marks include methylation and acetylation.  Other cellular processes such as post-transcriptional and post-translational modifications of non-coding RNAs, as well as transcription factors that can modulate gene expression, are also involved in epigenetics.

Epigenetic modifications are believed to be inherited during cell division, and to help in cell development and the creation of the hundreds of cell types in the human body, all carrying the same genes. Epigenetic mechanisms also provide an imprint of environmental influences on gene activity, which are passed on to future generations.

Epigenetic control is implicated in many diseases, including cancers and diabetes. Characterization of the possible mechanisms of DNA and histone modifications provides a detailed picture of disease-specific changes, and drug discovery is focusing on the proteins involved in these modifications, including readers, writers, and erasers.

I recently wrote a white paper “Introduction to epigenetics” and presented a webinar describing epigenetic proteins, and the use of isothermal titration calorimetry (ITC) as a biophysical tool to characterize the interactions between these proteins and histones and DNA.

The power of biophysics

Since chromatin binding and modifying activities are often found in multiprotein complexes, it is a challenge to describe individual chromatin-binding domains in vivo. To understand the function, activity, and specificity of chromatin-binding proteins, each interaction needs to be defined by a series of biochemical and biophysical in vitro assays. The dissociation constant (K_D) is an important parameter – the smaller the K_D, the tighter the binding affinity. ITC is a key assay to measure K_D values for the interactions between histone proteins or DNA with epigenetic proteins.  ITC provides quantitative K_D measurements, compared to biochemical binding assays (e.g  gel-shift assays, pull-down assays) which are more qualitative and can give yes/no for binding, and rank order affinities. The dissociation constants for chromatin interfaces are in the nanomolar (tight binding) to micromolar (weak binding) range, within the affinity range detectable by ITC; however, biochemical assays are not able to differentiate affinities in the low or high ends of this range. ITC can be performed with wild-type and mutant proteins, and histone peptides with different modifications to further characterize the selectivity and function.

ITC provides more information beyond the K_D, such as binding thermodynamics and binding stoichiometry. These measurements provide further insight into the specificity and selectivity of the interaction, the mechanism of binding, and the structure of the active binding domain. ITC is also used to evaluate binding of potential small molecule inhibitors during drug discovery.

Professor Andreas Ladurner, currently Professor and Chair of the Department of Physiological Chemistry at Ludwig-Maximilians-University in Germany, conducted pioneering epigenetics research as a postdoctoral fellow at University of California Berkeley. Using MicroCal ITC and X-ray crystallography, Prof. Ladurner’s group confirmed and characterized the interaction of a human bromodomain module to acetylated histone protein¹.  This was one of the first publications incorporating biophysics to characterize proteins involved in epigenetics.  Professor Ladurner uses MicroCal ITC in his current research on histone chaperones, proteins which interact with histones to control the assembly and reorganization of eukaryotic chromatin, and guide the assembly of nucleosomes².

The future of epigenetics

Several recent publications have shown data demonstrating epigenetics in vivo: in an article in Science³, scientists from the UK studied the effects of environmental stresses during early development, such as poor nutrition during pregnancy. The authors showed that protein restriction in mice from conception until weaning had a correlation to mouse growth restriction and DNA methylation at ribosomal DNA (rDNA). The rDNA methylation is an epigenetic response, and this modification remained into adulthood.

Four recent papers, in Nature Biotechnology and Nature Communications, validated the feasibility of epigenetic analysis for clinical applications, as well as the future of using epigenetics in design of medicines.

However, epigenetics is a controversial research area. To begin with, scientists have interpreted the meaning of epigenetics in different ways and there is not a universal consensus.  One meaning focuses on the molecular level of histone and nucleic acid modifications (marks) and the proteins involved in creating, recognizing, and removing these marks. Another perspective focuses on evolution and adaptation to biology, as well as what is called the “epigenome” and connecting epigenetics to specific diseases.

In the May 2, 2016 issue of The New Yorker was an article by Dr Siddhartha Mukherjee, presenting a personal view of epigenetics, discussing his mother and aunt ( identical twins) who had distinct personalities and life experiences. This article was adapted from Dr. Mukherjee’s new book The Gene: An Intimate History⁴. In several blogs posted after The New Yorker article was published, Dr. Mukherjee was criticized for only focusing on the chromatin epigenetic marks, and ignoring mechanisms of gene regulation such as transcription factors.  Dr. Mukherjee responded by publishing a point-by-point rebuttal online (since removed). In an interview with Nature, Dr. Mukherjee said he erred by omitting key areas of the science, but that he “didn’t mean to mislead.”

Some researchers argue that the in vivo experiments have been poorly designed, and it is not always possible for scientists to confirm that epigenetics is responsible for the observed effects.

To bring consensus in the epigenetics scientific community, a recent article⁵ defined many issues with current epigenetic and epigenomic studies, and proposed solutions that can be applied to epigenetics research and connection to diseases.

Epigenetics is an exciting field, and with proper attention to in vitro assays (including ITC) and in vivo experimental design, epigenetics can provide important insights into the molecular basis of diseases and how to treat them.

References:

1. Jacobson, et al, Science 288, 1422-1425 (2000)
2. Bowman et al. Nucleic Acids Res.  44, 3105-17 (2016)
3. Holland et al, published on-line July 7, 2016, DOI: 10.1126/science.aaf7040
4. Scribner, 2016
5. Birney, Smith and Greally, PLoS Genetics, 12(6): e1006105 (2016) doi:10.1371/journal.pgen.1006105

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