Introductory Chapter: Epigenetics and Optogenetics - The Science behind the Cover Blanket of Our Genome

1. Introduction

1.1 Epigenetics

For the longest time in the history of scientific research, a belief existed that DNA, the master molecule that makes up our genome, is the destination of living beings, the blueprint for every trait and disease that we might inherit or develop. Various landmark discoveries through many decades contributed to this “ultimate destination” tag of the DNA like the double helical structure in 1953 by Watson and crick, discovery of mutations in certain genes contributing to disease phenotypes such as phenylketonuria, cystic fibrosis, p53, and many more. These developments led to immense interest in the field and one of the most astounding accomplishments in this regard was the “human genome project,” which resulted in complete sequencing of the human genome. Soon after, complete genome sequences of closely related organisms and other model organisms were deciphered, published, and made available for use by every researcher across the globe. This led to the inception of the fields of bioinformatics and comparative genetics.

In the middle of all the euphoria about research on DNA and genes, it was being increasingly realized that only about 2% of the DNA in humans codes for proteins. The rest of the DNA was initially called junk DNA. However, an intriguing question surfaced regarding the reason for nature to preserve this huge amount (98%) of junk DNA if it did not serve any function. This seemed quite paradoxical to the concept of evolution.

This question paved the way for more research, and soon interest started booming in the field of epigenetics. This term has been used differently by different scientists from time to time, according to what could be proven using the resources and technology of that time. Epigenetics (from epigenesis) was first aimed to describe changes that take place when a zygote undergoes divisions and leads to differentiation (genesis) into different cell types, tissues, and organs. It was a beautiful concept to illustrate the differentiation potential of zygote, but the knowledge of the mechanisms responsible for this potential was lacking at that time. The term was originally coined by C.H. Waddington in 1942 as the phenomenon that changes the cells from totipotent state to fully differentiated state during embryonic development [1]. The phenomenon of heredity and the concept of genes were not known back then, and hence these definitions did not contain any molecular feature. Later, the term was defined by Riggs as “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” [2]. The most common definition of epigenetics today is “the study of phenomena that lead to heritable changes in gene expression without changing the sequence of nucleotides.” For the sake of simplicity and universality, an epigenetic trait was defined as “a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence” at the Cold Spring Harbor meeting in 2008 [3].

All these definitions were based on two important principles.

1. The change should influence gene expression and not DNA itself.

2. The change should be heritable.

With the discovery of histones, it was initially thought that these proteins only helped the DNA to wrap itself appropriately to fit into the nucleus. However, with advancing research, histones were viewed as the “interface” between DNA and the environment. These were the proteins that could change the accessibility of genes within the DNA to increase or decrease expression and interestingly, they could do it without the requirement to change the sequence of the underlying gene. This led to the identification of various histone modifications such as methylation, acetylation, phosphorylation, ubiquitylation, and so on, each one of them having their own kind of impact, that is, either increasing or decreasing gene expression. Research performed in the field also showed that different cells carry different combinations of histone modifications, and these combinations together constitute the histone code. More research on histones identified mechanisms such as histone sliding that can also influence gene accessibility and expression in response to various signaling pathways and at different stages of the cell cycle. DNA methylation on the 5′ cytosine also came to be recognized as a mechanism that could impact gene expression independent of the sequence of the gene that carries them.

Further research on model organisms was conducted on histones and DNA methylation to establish the transmissibility of epigenetic traits at the molecular level [4, 5]. One of the pioneering experiments performed on the mechanism of epigenetic inheritance was carried out by Manel Esteller and colleagues. The group extensively studied identical twins and verified that twin pairs that were older and/or had experienced different lifestyles had far greater differences in epigenetic marks (histone acetylation and DNA methylation) [6]. These studies were astounding as they helped in establishing the fact that DNA is not the destination to dictate all traits but patterns of expression and epigenetic changes can result in the establishment of different traits as a result of different environments, even in identical twins. Another study showed that supplementation of the diet of expectant mice with vitamin B, folic acid, choline, and betaine could alter the color of the fur of their offspring by affecting DNA methylation of the pigmentation genes [7]. Research on the agouti gene, which can cause diabetes and yellow color pigmentation of the fur in mice, has shown that offspring born to mice that were fed with supplements that resulted in methylation of the gene were slim and nondiabetic due to increased DNA methylation and consequent silencing of the agouti gene [8]. These experiments proved beyond doubt that we not only inherit our parents’ DNA but also their experiences and exposures, which influence our traits. Studies performed concomitantly and afterward also showed how the exposure of mothers to conditions such as smoking, alcohol consumption, stress during pregnancy, prenatal malnutrition, etc., can influence epigenetic patterns of key genes in offspring [9].

The very fact that epigenetic changes are heritable, yet reversible stimulated a lot of interest in the field because it provided a ray of hope to find a cure for many diseases that were initially thought to be terminal. This effect also impacts directly at the level of gene expression and hence can offer a lasting and more effective therapeutic approaches [10]. In addition, it established that different cells carry different epigenetic signatures, and that one cell type can be changed to another, or a diseased cell can be converted into a healthy one through changes in the epigenetic landscape.

More research identified more players of the field such as long non-coding RNAs, enhancer RNAs, micro RNAs, etc. It was in fact realized that the so-called “junk DNA” actually codes for these “regulator elements,” which play a role in regulating the expression of the genes that code for proteins [11]. Until now, we have been able to decipher very little information about epigenetic or regulatory elements. The fact that 98% of the genome codes for regulatory elements prompt us to believe that the field of epigenetics is very diverse and yet mostly unexplored. If this field is explored with the help of more advanced research tools and technology in the future, we might be able to find cures for many debilitating diseases of humans, might find more answers for our similarities and dissimilarities with other species, better understand evolution, and might develop a better understanding of the entire ecosystem by unraveling more connections related to gene–environment mechanisms. Increased knowledge of how gene–environment interactions operate acquired by means of increased knowledge of epigenetics through superior technology might answer many ecologically important questions for us and might enable us to understand the ecosystem and the role of Homo sapiens in this ecosystem in relation to other species and the environment more clearly and effectively.

2. Optogenetics

The sequencing of the genome in species as different as humans and plants has helped us to understand mechanisms of development, physiology, and evolution [12, 13, 14]. The field of epigenetics studies chemical modifications of the DNA as well as interactions that include genome-associated proteins to analyze differences in the expression of genes that are heritable and arise without a change of the DNA sequence. As such epigenetic mechanisms afford another mechanism of transcriptional control in regulating gene expression. While the field of epigenetics revealed an entire new layer of genetic regulation, optogenetics is the field that has allowed researchers to study cell signaling pathways and networks with unprecedented detail and resolution [15, 16]. This relatively new field exemplifies the power of taking a molecular approach to explore complex biological systems such as the brain in order to understand even the nature of emotions or psychiatric disorders [17]. Optogenetics is a combination of genetic manipulation and the use of optical tools. Genes that confer light responsiveness are inserted into cells of interest and allow for subsequent assessment of well-defined events in cells or even freely moving animals. Genetic tools allow the insertion of genes into cells that afterward respond to specific wavelengths of light. Subsequently, light can turn on or off specific signal cascades in cells and even trigger or inhibit the behavior of organisms. Thereby, optogenetics gives researchers an opportunity to obtain a deep view into an organism under optical control [18].

To understand the brain means to be able to reliably manipulate it and predict its response. Neuroscientists have long used electrophysiological techniques to stimulate particular brain areas or even single neurons [15]. Electrical stimuli activate neural circuitry, often without being able to stop neuronal activity. Neuropharmacological tools are based on drugs that are slow in their effects or not specific enough to stimulate individual cells. In 2005, a set of new techniques started to emerge that combined optical stimuli with genetic tools in order to control events in individual cells [19]. The field of optogenetics has since revolutionized experimental approaches to study cell signaling, metabolism, brain circuits, and organismal behavior.

Two pieces of information about the origin of the field are worth mentioning. As recounted by Karl Deisseroth [15], it was Nobel Laureate Francis Crick who suggested the creation of this new field in the late 1970s by stating that the major challenge facing neuroscience was the need to control one type of brain cell while leaving others unaltered. Later on, Crick proposed the use of light to achieve this control feat because it could be delivered in precisely timed pulses. The other piece of information relates to the fact that it was microorganisms that allowed optogenetics to come into existence. It had been known for many years that certain microorganisms generate proteins, which allow ions to cross the cell membrane in response to light. The genes coding for these proteins are known as opsins. One of the proteins, bacteriorhodopsin, discovered in 1971, is an ion pump that can be activated by photons of green light [20]. Later on, other opsins were identified, namely the halorhodopsins and channel rhodopsins, which are also light-gated ion pumps, more specifically, single-component light-activated cation channels. These discoveries have led to widespread use of optogenetic tools. Channelrhodopsin-1 (ChR1) and Channelrhodopsin-2 (ChR2) are found in the model organism Chlamydomonas reinhardtii. In 2005, several groups published the first accounts of using ChR2 as a tool for genetically targeted optical remote control, namely optogenetics, of neurons, neural circuits, and behavior of animals [19, 21, 22]. This marked the beginning of the field of optogenetics. Optogenetics has taken advantage of microbial opsins such as channel rhodopsin to genetically target and then remotely control excitable cells. In order to control cells or organisms, optical activation is superior to other methods because of its speed, ease of use, specific targeting, and precise temporal control of optical activation.

Acknowledgments

This publication resulted in part from research support to T.H. from the National Science Foundation [NSF IOS-1355034], Howard University College of Medicine, and the District of Columbia Center for AIDS Research, an NIH funded program [P30AI117970], which is supported by the following NIH Co-Funding and Participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, NIDDK, NIMHD, NIDCR, NINR, FIC, and OAR. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this chapter.