Until a few years ago, it was known that one of the principle chemical processes of epigenetics was to add a small chemical modification to genes. The process is called methylation, which adds a cluster (group) of methyl (an organic carbon, CH2) to the DNA base cytosine. The chemical name is 5-methylcytosine – 5mC. Adding 5mC to a gene turns it off; removing it turns the gene on. This is fundamental to the way cells dynamically alter the expression of DNA to adapt to the environment.

Just a few years ago it was discovered that there is another form of methylation, this time including a water-like molecule known as hydroxyl (OH). The name for this is 5-hydroxymethl-cytosine, the 5-hmC mentioned above. The take-home here isn’t the names or even the chemistry, but the fact this second form of methylation has been shown to be very important in how cells differentiate into different types. This is especially true for stem cells, which to various degrees are ‘blanks’ that epigenetic factors – likely through 5-hmC – are guided to differentiate into adult types of cells such as muscle or nerves.

While it required three paragraphs of preamble to get back to 5-mC and 5-hmC here is the real news: While scientists were aware of these two types of methylation, they had great difficulty in separating them within the cell and on DNA. That’s where the discovery of a new method for distinguishing both types during the sequencing of DNA comes in. The technique was developed in a collaboration between scientists at Cambridge University (Cambridge, UK) and Babraham Institute (Cambridge, UK), and published in the journal Science [26 April 2012, paywalled, Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution]. This work represents an important advance of genetic sequencing technology into the realm of epigenetics. By analogy, it is something like creating a new way of using a microscope through which the unknown is penetrated by a different perspective. While in one sense this tehcnique, which has the unmemorable name of oxidative bisulfite sequencing, is not ‘big news’ for the wider world, it could very well be one of those ‘keystone’ techniques that makes it possible to (eventually) answer questions such as “How do stem cells become adult cells?” or “How does the genetics of cancer cells go off the rails?” The field of epigenetics is in its infancy, this kind of advance in technique and technology gives it a lot more room to grow.

This new type of DNA is, for one thing, distinguished by existing outside the chromosome. Finding bits and pieces of DNA separated from the chromosome, in itself, isn’t too surprising. It’s a bit like finding flotsam along the shoreline; you expect some loose bits of material to be floating around in the cell. However, what scientists now call an extra-chromosomal circular DNA (eccDNA) may be something more significant.

One type of eccDNA, dubbed microDNA and recently discovered by scientists at the University of Virginia (USA) and the University of North Carolina (USA), is found in great numbers of relatively short strands (200-400 base pairs – the combinations of Guanine-Cytosine and Adenine-Thymine) in non-repeating sequences. Their finding has just been published in Science [08 March 2012, paywalled, Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues]. Where these ‘pieces’ of DNA come from has not been verified, but geneticists think it could be from cutting bits of chromosomal DNA (excision), replication of short DNA sequences, or reverse transcription of certain RNA. The research tends to show that microDNA mostly comes from deletions, which would indicate they are part of the repair and maintenance process for DNA.

The big question is what – if anything – are microDNA pieces for? Do they play an active role in the repair process, or are they the result (detritus) of that process? They do seem to be associated with gene variation between different types of cells. So far the researchers have found microDNA in human and mouse cells, but it may not be universal. At this point there are more questions than answers, although the pattern in genetic discovery tends to lead from the simple toward the complex. It is possible that microDNA and other eccDNAs have an important role in the genome – or not. It’s these kinds of questions that keep geneticists on their toes.

As is often the case with a medical advance surrounded with hype, there are doubts and concerns. A major new study by Johns Hopkins medical research in Science Translational Medicine [02 April 2012, paywalled, The Predictive Capacity of Personal Genome Sequencing] involving thousands of identical twins is outspoken about the failure of personal genomic analysis to identify a person’s risk for most common diseases. In fact, it flat out warns people not to uncritically accept negative genome test results.

Since the personal genome sequencing business is turning into a major growth industry and the stake of research on the links between genes and disease is enormous, this study is bound to be not only something of a knowledge-bomb, but also instantly controversial.

The basis of the study is recorded data on thousands of identical twins in the registries of Sweden, Denmark, Finland, and Norway along with the Twins Registry of the American National Academy of Science. Because identical twins are thought to share identical genomes, it should follow that if one twin presents a genetic based disease, statistics should indicate a prevalence of the same disease in the identical twin. The researchers used information on 24 diseases (cancer, autoimmune, cardiovascular, genitourinary, neurological and obesity-associated). Statistical models were created to predict the risk of each disease based on typical doctors’ diagnosis.

The results need a careful reading: A whole genome sequencing could indicate an increased risk of at least one disease, but most people would get negative test results for the majority of diseases in the study. Put another way, even for diseases with links to a genetic foundation, the presence of those genes in one individual is not a satisfactory predictor for another individual. Statistically, for example, if 2% of women show a genetic predisposition for ovarian cancer, it does not mean that the other 98% who test negative for the gene won’t get ovarian cancer.

You may have noticed that this is not a blanket denial of genomic analysis. The researchers are careful to say that genomic information can be very helpful for people with families who have a strong history of a particular disease. In addition, certain diseases that are shown to be particularly related to genetic variation such as coronary heart disease in men, thyroid autoimmunity, type 1 diabetes and Alzheimer’s disease are more likely to be accurately predicted by genomic analysis. This list is likely to grow as medical research advances.

However, in the broader perspective the ability of genomic analysis to predict a limited set of diseases leaves most people and most diseases unpredictable by this method. For example, while some hereditary cancers are gene influenced, hereditary cancer is rare. Most cancer is caused by genetic mutations acquired by environmental exposure, lifestyle choices (like smoking) and random errors in genes that occur during cell division. As one of the lead researchers, Bert Vogelstein of Johns Hopkins Kimmel Cancer Center (Maryland, USA) puts it:

We believe that genomic tests will not be substitutes for current disease prevention strategies. Prudent screening, early diagnosis and prevention strategies, such as not smoking and removing early cancers, will be the keys to cutting disease death rates.
[Source: EurekAlert]

First a little background: As a simple description, methylation is a switch mechanism for DNA. Chemically, methylation of DNA takes place when a methyl group (a molecule of carbon and hydrogen, CH₃) is added to either cytosine or adenine (two of the bases of DNA). The addition of a methyl group disables (turns off) the corresponding gene, that is, the gene is no longer available to guide the production of protein. Removing the methyl group, demethylation, reverses the process (turns it on). Methylation or demethylation is used within the genome to set up a pattern of active and inactive genes so that, among other things, cells become specialized. For example, a particular methylation pattern directs a cell to become a heart muscle. That pattern in then passed on (inherited) through subsequent cell divisions. The overall process is called epigenetic regulation and is one of if not the principle means of determining cell development, as in a stem cell that becomes a heart cell and stays that way through the life of an organism. It is also used to modify cell functioning in response to environmental conditions. As a rule, changes in epigenetic regulation are not inheritable through the egg or sperm (intergenerational), although the number of known exceptions is growing. The discovery and study of methylation and epigenetics is not much more than thirty years old with the bulk of the research starting in the 1990’s – it is a very young field.

Recent research in methylation continues to expand its reach. Neuroscientists primarily at the Johns Hopkins Brain Science Institute and led by Hongiun Song have been working on the role of methylation in the genome of brain cells (neurons). In previous research they had discovered that the brain cells of mice could be induced to faster growth through electric shock, which was decreasing the amount of DNA methylation. In the recent work, published in Nature Neuroscience [28 August 2011, paywalled, Neuronal activity modifies the DNA methylation landscape in the adult brain] they sequenced the genome of electrically stimulated and non-stimulated mouse brains and compared the results. They found that stimulated brains decreased or increased methylation (cytosine methylation) by 1.4%.

More significantly, they discovered that demethylation was taking place in neurons that were non-dividing on a large scale. This was surprising, as existing scientific consensus held that non-dividing cells were basically passive and the methylation would change very little, if at all. In other words, methylation in brain cells was almost exclusively for directing cell development.

“It was mind-boggling to see that so many methylation sites — thousands of sites — had changed in status as a result of brain activity,” Song says. “We used to think that the brain’s epigenetic DNA methylation landscape was as stable as mountains and more recently realized that maybe it was a bit more subject to change, perhaps like trees occasionally bent in a storm. But now we show it is most of all like a river that reacts to storms of activity by moving and changing fast.”

[Source: Johns Hopkins Medical Center]

Dr. Song’s expression is rather poetic, but the impact of this finding could be far-reaching. It implies that the methylation process in neurons has a function other than cell development (via cell division). What that function is and how exactly it works remains high on the research agenda. Since it is known that in other circumstances methylation is a means of responding to the environment, it is possible that a similar process is at work in the brain. I should mention that other studies have already forwarded the idea that methylation, or some form of epigenetics, may be the basis of memory.

The idea behind this particular nanosensor came from study of natural biosensors within cells. All living things monitor their condition, from the largest scale of organs to the smallest nanoscale chemistry of individual cells. At the level of the cell, there are billions of specialized proteins or RNA that perform the task of a sensor by reacting to the presence of very specific molecules. For example there are many loops or cyclical chemical pathways, where a certain condition, say a need for energy, triggers a chemical and physical change in one sensor protein. It in turn signals for production of more energy. When enough energy is produced, another sensor protein accumulates to the point where it turns off energy production.

Scientists at the University of California, Santa Barbara (USA) and the University of Rome Tor Vergata wanted to emulate this natural sensor-signal process with a specific target in mind. As published in the Journal of the American Chemical Society [04 August 2011, paywalled, Transcription Factor Beacons for the Quantitative Detection of DNA Binding Activity] they developed a sensor made from DNA that becomes luminescent (glows) when it encounters a particular protein of the type called a transcription factor. These are proteins used by cells to control the production of molecules (usually other proteins). There are literally thousands of transcription factors, but when scientists ‘reprogram’ cells for example in stem cells; they often change only a handful of factors. The trick is to know whether the reprogramming has worked properly or not. That’s where the nanosensors come in.

There are many techniques for reading transcription factors; most of them require laborious separation of specific proteins and examination either under microscopes or with chemical detectors. As one of the researchers put it, “With the new sensors, we just mash the cells up, put the sensors in, and measure the level of fluorescence of the sample.”

The sensors are built by re-engineering three natural DNA sequences, each set to recognize a different transcription factor, by adding a molecular switch that becomes fluorescent when activated. Eventually this technique can be extended to thousands of transcription factors. In turn, the technique can help scientists and doctors monitor the level of drug activity, screen for certain kinds of cancer signaling proteins or any other application where transcription factors might reveal an underlying biological condition. In short, this technique could be very useful and practical.

The technique also seems relatively simple, but it will ultimately compete with many other technologies (sometimes called assay technology) that read the presence of transcription factors and other protein signaling molecules. It’s a burgeoning field of cell biology and of sensor technology-in-the-very-small.

When the Broad team discovered more than 3,500 unique lincRNAs in the human and mouse genomes in 2009, “the potential was enormous, and we wanted to know what they could be doing.”

[Source: Technology Review]

Here’s the scenario: A team of researchers at the Broad Institute (a joint operation of Harvard University and Massachusetts Institute of Technology, USA) discovered in 2009 that human and mouse genomes were encoded to produce thousands of a hitherto unknown form of RNA. The role of RNA, as commonly understood, is to carry the genetic code for protein production from the DNA to the locations of protein manufacture. Over the years, however, new forms of RNA were discovered – microRNA, mRNA, siRNA, RNAi, among others – and the range of function for RNA extended. In fact, the team at Broad Institute had discovered a form of RNA that didn’t appear to have any role in the coding and manufacturing of proteins. Called a lincRNA for ‘large intergenic non-coding RNA’, this form of RNA was found in all cells in great numbers. The team eventually identified over 3500 unique forms of lincRNAs. The question staring them in the face and making their adrenalin pump (so to speak) was, of course, what do all these lincRNAs do?

It turns out that at least one of the things lincRNAs do is coordinate and organize the assembly of proteins in embryonic stem cells – and probably all other cells as well. This is something of a revelation. It has always been thought that proteins themselves direct the development of cells, now it appears that lincRNAs provides the structure – a kind of chemical scaffold – on which cell protein is assembled. In stem cells, it is the role of lincRNAs to provide the all-important control of whether a stem cell remains pluripotent (able to turn into almost any other kind of cell) or differentiates into a specific type of adult cell. This role is so important to the development of life that discovery of a previously unknown agent of such influence as lincRNA is almost shocking. Except that for biochemists this is the kind of thing that makes a career, or even a Nobel.

The researchers at Broad Institute, with first author Mitchell Guttman and senior staffers David Root and Eric Lander, decided in 2009 to concentrate on the activity of lincRNA in embryonic stem cells, which are heavily studied and of such obvious biological importance. In order to determine the role of lincRNA, they used genetic techniques to turn off and on the production of specific lincRNAs. Eventually they isolated about 100 forms of lincRNA that appeared to be at work in stem cells. From there they used biochemical analysis to follow the effect of lincRNA on cell protein.

The results of their work, published in the journal Nature [28 August 2011, paywalled, lincRNAs act in the circuitry controlling pluripotency and differentiation] is the first comprehensive view of lincRNA at work in a specific cell type. As one of the researchers put it, “lincRNAs are like team captains, bringing together the right [protein] players to get a job done.”

Sports analogies aside, the discovery of a whole new class of RNA – one with such a powerful role in the development of cells – opens the way to explore yet another massive complication in the processes of life. This might be overstating the case, but probably not. In any case, this is the kind of challenge that scientists live for, which is definitely not an overstatement. The opportunity to experiment and provide answers to big questions (even if the subject matter is very small) is rare enough.

I can see the “?” form over your head. I’ll explain in a bare moment, but first the “Why?” Using the new DNA, biochemists will be able to create proteins that mimic disease conditions, or components of diseases – and turn them on or off as part of experimental testing. Doing this should give scientists much greater insight into the role of proteins in diseases and how to control them. In short, it creates a kind of ‘sandbox’ (controlled) environment to test hypotheses about diseases and how they work. Now, for a bit of explanation…

The place to start is with the concept of phosphorylation. This is what happens to proteins when a phosphate group (PO4) is added, it activates (or deactivates) the protein (often an enzyme) like a chemical on-off switch. Phosphorylation is one of the most fundamental of biochemical processes, used within cells for an untold number of chemical pathways, and the subject of an enormous amount of research. Interestingly, although phosphorylation is crucial to the timing and control of many biological processes, it is not something directly controlled by DNA. It falls into the area known as epigenetics, genetic (reproducible and sometimes inheritable) characteristics that occur largely outside of the usual DNA/RNA mechanics.

What the Yale researchers did is take the phosphorylation capability and code it into the genome, into the DNA of E. coli, a remarkable technical feat. They added the ability to synthesize phosphoserine, a key compound in the phosphorylation of enzymes (among other things), and added it to the natural bag of genetic tricks – using synthetic biology to produce a new form of natural biology (so to speak).

The outcome of adding new DNA to E. coli is that the bacteria can produce ‘natural’ proteins, proteins that scientists choose to create, that have the phosphorylation capability built into them.

“Essentially, we have expanded the genetic code of E. coli, which allows us synthesize special forms of proteins that can mimic natural or disease states,” said Jesse Rinehart co-corresponding author of the research. “What we have done is taken synthetic biology and turned it around to give us real biology that has been synthesized.”

[Source: EurekAlert]

This does sound like a new sandbox to play in, but there are probably rules and limitations built into this sandbox that are yet to be discovered. I’m using the sandbox analogy to emphasize that while the ability to synthesize phosphorylate proteins via DNA is a powerful concept, it may yet prove to have problems in practice. It’s certainly possible that synthesizing disease proteins with phosphorylation built-in is a key to understanding the interaction of living proteins and disease conditions. However, there is more – much more – to the complexity of biological processes that is not well understood, especially in the area of epigenetics. A few years down the road, and the techniques used in this research may be deemed ‘flawed’ or ‘too difficult’ or any number of other conditions that will sideline the technique. Or this could be the beginning of a highly successful approach that opens the research doors to the understanding of many forms of disease. Hopefully so.

The next steps for the Yale team is to actually manufacture proteins for diseases such as cancer(s), type 2 diabetes and hypertension (for example) and put them into experimental environments where they can be observed in vivo or in vitro.

Epigenetics is a relatively new field, one that continually produces surprises for geneticists and biochemists. It tends to provide challenging contradictions to the notion that genetic responses take place over millennia due strictly to DNA mutation and evolutionary pressures. Epigenetics started with showing how responses to immediate environmental conditions such as drought, famine or stress can be incorporated into the development of an organism. Now it’s showing that some of these responses can be carried into genetic inheritance. Put another way, this is a form of genetic response that can occur within a single generation and is not necessarily highly conditioned by the usual factors of evolutionary selection.

The research by Howard and Dean worked with plants that showed a marked degree of response to cold weather – flowering only at exactly the right time and conditions. These plants have a gene, named FLC, that is related to flowering by ‘remembering’ the length of preceding cold seasons. By using mathematical modeling and experimental analysis, they were able to show that FLC is either on or off, and that after a cold period a high proportion of FLC genes are turned off. This condition delays flowering. In this case further testing indicated that the FLC gene was turned off by selective positioning of the histones modified during a cold period. This epigenetic memory can be passed to succeeding generations of the plant.

This is one example of epigenetic modification of genetic properties. There are many others, such as the recent studies showing that methylation of genes (attachment of a methyl group to a cytosine nucleotide) can also result in turning genes on and off in a heritable fashion. For example one study by Meyer and Elbert (University of Konstanz, Germany) indicates that in response to high stress levels in pregnant mothers, methylation of various genes can be carried to the baby and may express itself in a variety of disorders. Economist [21 July 2011 A mother’s stress while she is pregnant can have a long-lasting effect on her children’s genes]

[SciTechStory: Epigenetics and methylation: New DNA bases linked to protein]

It seems pretty obvious that these new bases 5 through 8 are not replacements for the more common four. So what’s the deal, why are these unmemorable variants of cytosine important?

They are the result of a process called methylation. In DNA methylation is a chemical process that adds an organic molecule, a methyl group with a basic formula of CH₃, to the base cytosine. When a methyl group is tacked onto a nucleotide, it changes its characteristics, namely the configuration or shape. Simply put, it causes that portion of the double helix to fold into itself. This shields the underlying nucleotide from activation – in short, it’s turned off. Most of the human chromosome available for methylation has been turned off in this way. Where they are not turned off, that’s where a very large percentage of genes are ‘expressed’ – involved in producing protein.

What genetic scientists are discovering is that while DNA may provide the blueprint or basic instructions for building proteins, which genes are involved at what specific time and in what specific ways is often the result of the process of methylation. Methylation is how living cells respond to the environment. For example, a response to stress conditions is represented by changing patterns of methylation, turning genes on and off. These changes are then copied when new cells are made. It’s a process studied as the relatively new field of epigenetics.

What the Zhang team discovered is that a particular protein group called Tet is responsible for the conversion of cytosine in a nucleotide into 5-methylcytosine and then the other three methylated bases. While the details of how this works are still part of the ongoing research, it seems likely that the interaction of Tet proteins with DNA is a key element in the methylation process.

This makes the Tet proteins a potentially important subject for genetic and medical research. Someday, though not soon, the process of methylation through Tet proteins will be better understood. It may be possible to use Tet protein in some form to control the methylation of genes (turning them on or off). It would be hard to overestimate how powerful (and dangerous) a tool this might be, for it could be a pathway to controlling epigenetics. This could include countering the environmental causes of cancer or reprogram adult cells into stem cells.

That potential is still far away, but with this advance in the understanding of DNA methylation scientists move closer to understanding the mechanics of epigenetics.

[SciTechStory: Part of what makes us human may be what’s missing]
[SciTechStory: Small steps toward understanding the epigenome]

This ‘swapping of bases’ is a trick that nature might have done. Last year (2010) there was a relatively well publicized rhubarb among scientists about the discovery of arsenic life by a team of NASA funded researchers. They believe(d) they found a strain of bacteria living at the bottom of Mono Lake in California that due to a lack of phosphorus had substituted arsenic for phosphorus in key biological compounds (not in DNA but in ATP). [SciTechStory: An odd couple: Arsenic and life] The claim for arsenic life did not hold up too well under close scrutiny, but the mechanism at work, an evolutionary chemical substitution, is relevant to the current story.

In this case, an international group of researchers (Germany, USA, France, Belgium) looked at the structure of DNA and decided that if any base could be substituted, it would be thymine. (RNA already uses uracil instead of thymine.) They reckoned that 5-chlorouracil was chemically and structurally close enough to thymine to – perhaps – be taken up by DNA. Thus they began their experiments with the labster’s favorite bacteria, E. coli, by essentially putting it on a diet of nutrient spiked with chlorouracil and continually lowering the amount of thymine available.

As they reported in the journal Angewandte Chemie International Edition [27 June 2011, paywalled, Chemical Evolution of a Bacterium’s Genome] the substitution was accomplished by chemical evolution, where the absence of thymine caused the E. coli to start using chlorouracil and those bacterium that did thrived. This was evolutionary pressure at work, only with E. coli, which has a procreation cycle of about 4 hours, things happen relatively fast. The process was aided by the latest in automated monitoring and nutritional equipment so that the mix of chlorouracil and thymine was modulated to keep the E. coli population from declining too far but keep the pressure on to adapt to a chlorouracil-rich environment.

Within 23 days it was apparent that E. coli was taking up the chlorouracil replacement and the mix was accelerated. By 140 days the bacteria had substituted about 90% of its DNA with chlorouracil instead of thymine. The E. coli still require some thymine to live, but many interesting things happened to the DNA: The E. coli could successfully switch between thymine and chlorouracil, a number of chlorouracil mispaired with guanine, and in some cases the entire DNA structure was rearranged. As the researchers said, “It would have been impossible to predict the genetic alterations underlying these adaptations from current biological knowledge…”

E. coli with a 90% swap for chlorouracil is not a new life form; it still needs thymine to stay alive. However, it is a big step in that direction. Of course, as in so much of life, it’s the last step that’s the hardest. To finally produce a life-form that requires a different chemical for a DNA base has enormous implications. It would open the door to creating new organisms with all kinds of altered chemistry, while at the same time providing a built-in incompatibility with existing life. (We hope.)

For a more detailed description, check out Derek Lowe’s blog. In the Pipeline: A first step toward a new form of life