̽��ֱ�� of Cambridge - methylation

How large is the alphabet of DNA?

sc604 — Thu, 12 Dec 2013 09:22:38 +0000

̽��ֱ��mechanisms which cause certain genes to be switched on or off, and are thought to play a role in cancer development and stem cell differentiation, can now be accurately detected and studied thanks to a new DNA sequencing method.

̽��ֱ��technology developed by Cambridge Epigenetix is helping researchers understand modifications to DNA, by detecting ‘extra’ DNA bases, which until now could not be definitively identified.

There are four standard DNA bases (Guanine, Cytosine, Adenine and Thymine), and the way they are ordered determines the makeup of the genome. In addition to G, C, A and T, there are also small chemical modifications, or epigenetic marks, which affect how the DNA sequence is interpreted and control how certain genes are switched on or off. ̽��ֱ��study of these marks and how they affect gene activity is known as epigenetics.

̽��ֱ��most-studied mark is 5-methylcytosine (5mC), which is formed when molecules of methyl attach to the cytosine base of DNA, a process known as methylation. In 2009, a ‘sixth’ base, 5-hydroxymethylcytosine (5hmC) was discovered in human DNA, and subsequently two further modified DNA bases, 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) were also identified.

Professor Shankar Balasubramanian of the Department of Chemistry founded Cambridge Epigenetix in 2012 to develop innovative epigenetic research tools that can identify, decode and help elucidate the function of the ‘extra’ DNA bases.

Standard DNA sequencing methods work by reading the features of the four standard bases, but cannot detect whether a cytosine base has been methylated. In order to address this shortcoming, a method called bisulfite sequencing was developed to detect methylation by adding a bisulfite reagent that converts the non-methylated cytosine bases to uracil, one of the subunits of RNA. By sequencing bisulfite-treated DNA, researchers can identify which cytosine bases were originally methylated and which were not.

However, because 5hmC and 5mC are both resistant to bisulfite treatment, it is impossible to distinguish between these two epigenetic marks using traditional bisulfite sequencing.

̽��ֱ��reason this is a key distinction to make is that 5mC and 5hmC are thought to have completely different physiological functions. Research on the link between gene expression and methylation indicates that there are certain sites where methylation causes the gene to be switched off and silenced, whereas hydroxymethylation causes the gene to be switched on.

“Functionally, they have profoundly different meanings, yet we haven’t been able to tell the difference between them using typical sequencing methods,” said Professor Balasubramanian.

Following the discovery of the fifth and sixth bases, Professor Tony Green from the Department of Haematology encouraged Professor Balasubramanian to think about a new method of sequencing to detect these modifications. Balasubramanian and his PhD student Michael Booth co-invented such a method, known as oxidative bisulfite sequencing.

Oxidative bisulfite sequencing allows researchers to quantitatively measure 5mC and 5hmC at single-base resolution, enabling more accurate DNA sequencing.

̽��ֱ��technique works by chemically oxidising 5hmC to 5fC, which like cytosine is susceptible to bisulfite treatment. Once the oxidative bisulfite reaction is complete, 5hmC and cytosine will appear in the sequence as thymine, so that the only cytosine bases remaining in the sequence are truly 5mC.

“In one reaction, you can get an accurate representation of methylation without having to factor in the ‘contamination’ from hydroxymethyl C,” said Professor Balasubramanian. “What our research group and Cambridge Epigenetix are doing is bringing this capability to go beyond the standard four letters of the genetic alphabet in a way that benefits from all the general innovation brought from ‘next generation’ sequencing technology, such as the Solexa/Illumina approach.”

Research studies indicate that dynamic regulation of DNA function by these epigenetic marks is essential for normal foetal development and plays an important role in cancer, neurological disorders and other diseases. In addition, it is thought that DNA modification plays a central role in stem cell reprogramming.

“Reprogramming the way DNA functions is fundamental to all living systems,” said Professor Balasubramanian. “It’s remarkable that for so long, we weren’t aware of these other modifications in human DNA. If we’ve found four more bases since 2009, then who are we to argue that nothing else is there?”

Cambridge Epigenetix conducted an alpha trial of its first product, TrueMethyl, late last year, and a beta trial in 13 laboratories around the world earlier in 2013. TrueMethyl is available worldwide, and the company has a range of other epigenetic research tools currently in development.

̽��ֱ��company is funded by Cambridge Enterprise, the ̽��ֱ��’s commercialisation arm, and Syncona Partners.

New sequencing technology is transforming epigenetics research, and could greatly improve understanding of cancer, embryo formation, stem cells and brain function.

It’s remarkable that for so long, we weren’t aware of these other modifications in human DNA. If we’ve found four more bases since 2009, then who are we to argue that nothing else is there?

Shankar Balasubramanian

Waugsberg via Wikimedia Commons

Queen bee larvae in royal jelly. Worker bees and the queen have exactly the same DNA sequence, but queen larvae are fed royal jelly which epigenetically modifies their DNA so they grow to be larger and fertile.

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Exposure to micronutrients prior to pregnancy has been associated with gene modifications in offspring

gm349 — Wed, 22 Feb 2012 11:02:59 +0000

̽��ֱ��offspring of women who were given micronutrient supplements (minerals needed in small quantities, such as iron, iodine and vitamin A) before they became pregnant had gene modifications at birth as well as when they were tested at 9 months.

̽��ֱ��changes to the genes, called methylation, have previously been associated with the development of the immune system, although this study did not provide direct evidence that the activity of these genes has changed. ̽��ֱ��research, funded by the BBSRC, was published today in the journal Human Molecular Genetics in advance online publication (DOI number DDS026).

Professor Nabeel Affara, lead author of the study from the ̽��ֱ�� of Cambridge, said: “ ̽��ֱ��mechanism by which micronutrients influence methylation changes is still to be worked out, but it is known from other work that the genes of the immune system undergo such changes as immune function develops, particularly in early postnatal stages and early childhood.

“These changes are part of the normal development of the immune system provided adequate nutrition is available. Where this is not the case, different patterns of methylation may occur, altering the activity of key genes and therefore potentially the effectiveness of the immune system. ̽��ֱ��result is likely to be reduced ability to fight infection and hence susceptibility to infectious diseases.”

̽��ֱ��study used DNA samples from a Medical Research Council (MRC) micronutrient supplementation trial where women attempting to get pregnant are given either a cocktail of micronutrients or a placebo until pregnancy is confirmed (approximately an 8 weeks period). ̽��ֱ��research was conducted in ̽��ֱ��Gambia where there is seasonal variation in the availability of micronutrients with an alternation between the dry season (when they harvest and food is plentiful) and the wet season (when there is less food available and therefore poorer nutrition). Individuals born in the wet, nutritionally poor season have been found to be more susceptible to infection.

Professor Affara added: “This has huge public health implications for regions of the world where food security is an issue. If we have an improved understanding of what nutrition is important and the mechanisms by which this important environmental factor interacts with gene function, we can target nutritional intervention to improve health in later life.”

Scientists find that micronutrients affect methylation, which has been associated with changes in the immune system.

̽��ֱ��mechanism by which micronutrients influence methylation changes is still to be worked out, but it is known from other work that the genes of the immune system undergo such changes as immune function develops, particularly in early postnatal stages and early childhood

Professor Nabeel Affara, lead author of the study from the Department of Pathology

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Rainy season in ̽��ֱ��Gambia

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Environment and Diet leave their prints on the heart

bjb42 — Tue, 29 Nov 2011 12:00:23 +0000

̽��ֱ��new data collected greatly benefits a field that is still in its scientific infancy and is a significant leap ahead of where the researchers were, even 18 months ago.

Researcher Roger Foo explains: “By going wider and scanning the genome in greater detail this time - we now have a clear picture of the ‘fingerprint’ of the missing link, where and how epigenetics in heart failure may be changed and the parts of the genome where diet or environment or other external factors may affect outcomes.”

̽��ֱ��study originally began investigating the differences in DNA methylation found in the human heart. Researchers compared data from a small number of people with end-stage cardiomyopathy who were undergoing heart transplantation, and the healthy hearts of age-matched victims of road traffic accidents.

DNA methylation leaves indicators, or “marks”, on the genome and there is evidence that these “marks” are strongly influenced by external factors such as the environment and diet. ̽��ֱ��researchers have found that this process is different in diseased and normal hearts. Linking all these things together suggest this may be the “missing link” between environmental factors and heart failure.

̽��ֱ��findings deepen our understanding of the genetic changes that can lead to heart disease and how these can be influenced by our diet and our environment. ̽��ֱ��findings can potentially open new ways of identifying, managing and treating heart disease.

̽��ֱ��DNA that makes up our genes is made up of four “bases” or nucleotides – cytosine, guanine, adenine and thymie, often abbreviated to C, G, A and T. DNA methylation is the addition of a methyl group (CH3) to cytosine.

When added to cytosine, the methyl group looks different and is recognised differently by proteins, altering how the gene is expressed i.e. turned on or off.

DNA methylation is a crucial part of normal development, allowing different cells to become different tissues despite having the same genes. As well as happening during development, DNA methylation continues throughout our lives in a response to environmental and dietary changes which can lead to disease.

As a result of the study, Foo likens DNA methylation to a fifth nucleotide: “We often think of DNA as being composed of four nucleotides. Now, we are beginning to think there is a fifth – the methylated C.”

Foo also alludes to what the future holds for the study: “...and more recent basic studies now show us that our genome has even got 6th, 7th and 8th nucleotides... in the form of further modifications of cytosines. These are hydroxy-methyl-Cytosine, formylCytosine and carboxylCytosine = hmC, fC and caC! These make up an amazing shift in the paradigm…”

As in most studies, as one question is resolved, another series of mysteries form in its place. ̽��ֱ��study shows that we are still on the frontier of Epigenetics and only just beginning to understand the link between the life we lead and the body we have.

A ̽��ֱ�� of Cambridge study, which set out to investigate DNA methylation in the human heart and the "missing link" between our lifestyle and our health, has now mapped the link in detail across the entire human genome.

By going wider and scanning the genome in greater detail this time - we now have a clear picture of the ‘fingerprint’ of the missing link, where and how epigenetics in heart failure may be changed and the parts of the genome where diet or environment or other external factors may affect outcomes.

Roger Foo

Greencolander from Flickr

Candy Hearts

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Scientists find "missing link" between heart failure and environment

bjb42 — Wed, 13 Jan 2010 00:00:00 +0000

̽��ֱ��Cambridge team compared heart tissue from two groups - patients with end-stage heart failure and those with healthy hearts. ̽��ֱ��diseased tissue came from men who had undergone heart transplants at Papworth Hospital, Cambridge, and the healthy hearts from age-matched victims of road traffic accidents.

They found that specific regions of the DNA in the diseased hearts contained "marks" known as DNA methylation, whereas the healthy hearts did not. This is the first study linking DNA methylation with human heart failure.

DNA methylation is already known to play a key part in development of most cancers, and its role in other complex diseases such as schizophrenia and diabetes is being investigated.

This study, funded by the British Heart Foundation, suggests the process also underlies development of different types of heart disease.

According to lead author Dr Roger Foo of the ̽��ֱ�� of Cambridge: "DNA methylation leaves 'marks' on the genome, and there is already good evidence that these marks are strongly influenced by environment and diet. We found that this process is different in diseased and normal hearts. Linking all these things together suggests this may be the 'missing link' between environmental factors and heart failure."

̽��ֱ��findings deepen our understanding of the genetic changes that can lead to heart disease, and how these can be caused by diet and the environment. As a result, Foo's findings should open up new ways of managing and treating heart disease.

" ̽��ֱ��next stage of our research is to find hotspots in the genome. This should help us identify people at risk of heart disease, and pinpoint patients whose disease will progress fastest. This would radically alter how we manage patients with heart disease, allowing us to target treatments and tailor monitoring," Foo explains.

̽��ֱ��DNA that makes up our genes comprises four "bases" or nucleotides - cytosine, guanine, adenine and thymine, commonly abbreviated to C, G, A and T. DNA methylation is the addition of a methyl group (CH3) to cytosine.

When bound to cytosine, the methyl group sticks out. This means it looks different and is recognised differently by proteins. As a result, methylation alters how genes are expressed, ie which are turned on and off.

Foo likens DNA methlyation to a fifth nucleotide: "We often think of DNA as being composed of four nucleotides. Now, we are beginning to think there is a fifth - the methylated C."

DNA methylation is a crucial part of normal development, allowing different cells to become different tissues despite having the same genes. As well as happening during development, DNA methlyation continues throughout our lives in response to environmental changes and can lead to disease.

According to the study's first author, Dr Mehregan Movassagh of the ̽��ֱ�� of Cambridge: "DNA methylation is a mechanism by which the environment and diet alters the expression of certain human genes, and has been the explanation for why, for instance, identical twins may have differing features and differ in their susceptibility to disease, despite having an identical set of genes."

It is also a very widespread process, occurring in plants and insects as well as vertebrates. In honey bees, for example, it is the reduction in DNA methylation that occurs as a result of feeding royal jelly which causes genetically identical larvae to develop into a queen, rather than a worker.

Epigenetic factors, such as DNA methylation, are currently the focus of much medical research because they offer further insight into disease than simply looking at our genes.

"We already know that several genes play an important role in heart failure. Researchers have looked at mutations in these genes and sometimes don't see any, so it could be methylation, not mutation, which is responsible for the altered expression that leads to disease. This opens a new window on the link between genome and disease," Movassagh says.

Professor Jeremy Pearson, Associate Medical Director at the British Heart Foundation (BHF), which funded the research, said: "By detecting these molecular changes in failing hearts, this research suggests that previously unsuspected mechanisms contribute to the development of heart failure. ̽��ֱ��findings open up the possibility of identifying new ways to treat this debilitating condition, which affects more than 700,000 people in the UK. We're supporting these researchers and others around the country to help us turn these vital discoveries into treatments for patients."

̽��ֱ��research is published in PLoS ONE.

Scientists have found what they believe is the missing link between heart failure, our genes and our environment. ̽��ֱ��study could open up completely new ways of managing and treating heart disease.

We often think of DNA as being composed of four nucleotides. Now, we are beginning to think there is a fifth - the methylated C.

Dr Roger Foo

Mykl Roventine from Flickr

February is American Heart Month

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