- Tet1 Enzyme Based Enrichment Method for Methylome Sequencing: TamC-Seq
- Introducing Aba-seq for Enzyme Based High-Res Mapping of Mammalian Hydroxymethylomes
- Methylome Data in Lethal Prostate Cancer Supports Personalized Medicine
- New Years Resolution, Reflection on Cancer Research
- Did Epigenetics Make Us Smart?
- Bill Graham on Sirtuin3 Reprograms Mitochondrial Epigenetic Pathways: How Diet Affects Age
- Doug on Will the Long History of Breast Cancer Research Culminate with Epigenetics Based Personalized Medicine?
- Canada Joins the International Human Epigenome Consortium – Q&A with Tomi Pastinen of Génome Québec | Epigenetics Experts Blog on Q&A with BLUEPRINT’s Henk Stunnenberg on the New Leukemia, Blood Epigenome Project
- Doug on Oxidative Bisulfite Sequencing (oxBS-Seq) A Brilliant Advance for Epigenetics
- The Epigenetics of Real-Life Stress and Serotonin | Epigenetics Experts Blog on Situational Stress Makes Short-Term Epigenetic Changes
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First of all, a hearty congratulations to Dr. Shinya Yamanaka and Dr. John Gurdon for winning this year’s Nobel prize for Medicine, for their discoveries that adult cells could be transformed back to embryonic-like states. Recently, Dr. Yamanaka has publicly warned of dangerous “stem cell therapies” currently offered in various countries, without any pre-clinical testing in animals. This was an important message considering possible tragedies, both for any patients desperate for a cure, who end up sick or dead…and for the public, who might lose their trust in potential future stem cell therapies developed safely under strict scientific methods.
Induced pluripotent stem cells (iPSCs) can be transformed from somatic cells, through the expression of only four transcription factors, using Kyoto University viral delivery methods. However, its been shown that the process of reprogramming these cells results in genetic and epigenetic aberrations in comparison to embryonic or parental somatic cells. Other methods also have been developed, or are in development, to produce IPSCs. For example protein-induced pluripotent stem cells. Goals for any method include producing non-tumorigenic, completely reprogrammed cells
For the recent pub, Sergio Ruiz & Dinh Diep et al. Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells. (2012) PNAS, the authors developed a method to discriminate between hESCs and hiPSCs based on targeted bisulfite sequencing with padlock probes, to produce unbiased hierarchical clustering of global methylation. Interestingly, higher reprogramming efficiency was associated with less difference in methylated CpG sites between somatic and iPSC cells. Methylation changes at only nine genes amongst all the iPSC cell lines, regardless of origin cell type. The epigenetic signature for segregation was made clear because areas of lower CgP content were covered. Previous analysis by restricted representation bisulfite sequencing (RRBS), which focuses on CpG islands, could not differentiate between ESCs and iPSCs in this way. The caveat is that more cell lines need to be tested to enhance this epigenetic signature’s utility.
Ruiz S, Diep D, Gore A, Panopoulos AD, Montserrat N, Plongthongkum N, Kumar S, Fung HL, Giorgetti A, Bilic J, Batchelder EM, Zaehres H, Kan NG, Schöler HR, Mercola M, Zhang K, & Izpisua Belmonte JC (2012). Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America, 109 (40), 16196-201 PMID: 22991473
Sure, M.D.s often suffer a lot of pressure. But as I learned in a brief hospital job, nurses really bear the brunt of all the biological clean-up, red tape, weird hours, patient complaints, and snippy doctors’ demands. So this new study in PLoS One on stress-related epigenetic changes in shift-working female nurses really caught my attention, and seemed like a good followup that post on situational stress and epigenetics. Nurses under high stress appear to have their gene expression epigenetically regulated in a way that may decrease serotonin in the brain’s synapses. It seems a bit like the reverse of Prozac, and it bears a passing resemblance to what might happen at the beginning of depression.
By interfering with serotonin transporter proteins, Prozac and other SSRI drugs allow the “well-being” neurotransmitter serotonin to exert its effects on recipient neuron for a little longer. But according to this study by Jukka Alasaari and Tiina Paunio at the National Institute for Health and Welfare in Helsinki, Finland — and colleagues — high stress appears to trigger less promoter methylation of the serotonin transporter gene, SLC6A4. That usually means cellular machinery makes more mRNA from a gene, and as a result, expresses it more fully. So: more stress, more serotonin transporter, less serotonin the synapses.
I’d expect our physiology to compensate stress, but that’s often not how it works. From the authors’ citations, here’s another example of high stress leading to low serotonin transporter-gene methylation. Alasaari and Paunio discuss it this way:
In the context of this study, we hypothesize that hypomethylation of SLC6A4 presents an adaptational mechanism for stress. While this adaptation is physiological and initially serves to maintain the individual’s best possible functional capacity during stress, it might, eventually, increase risk for functional disturbances, such as decreased cognitive ability and depressed mood, simultaneously with failure of other coping mechanisms. Interestingly, relative lack of serotonin in brain is one of the major hypotheses of depressive disorder, and serotonin transporter is known to be one of the major targets of many antidepressants, including the selective serotonin re-uptake inhibitors .
So maybe we’re talking about the first steps in the route to disease, although I don’t understand how increased serotonin re-uptake is helpful when you’re stressed.
But back to the experiments themselves. The authors chose the high- and low-stress extremes from among a large (n=5615) group of nurses who answered questionnaires based on Karasek’s Model, in which people in high-stress environments report high work demands and low control over the situation, while people under low stress report low demands and high control. The 49 remaining nurses also answered surveys about burnout and non-clinical depression, with about half of the high-stress group showing signs of burnout and at least mild depression. Not surprisingly, the low-stress group displayed few such signals — only two people with mild depression.
Thereafter, the researchers used bisulfite sequencing to test the nurses’ blood for the methylation of five CpG sites in the serotonin transporter’s promoter. And after a fair amount of statistical work, discovered a significant association between the high-stress group and low promoter methylation.
But oh no. We’re talking about testing blood, and we’re speculating about things that happen in the brain. And so it’s time to talk about caveats. But at least the authors were nice enough to mention the same ones I came up with.
The research relies on peripheral blood leukocyte samples as a proxy for neurons, which are ethically unreachable. (In Soviet Union, proxy relies on you.) No one really knows if the gene methylation in these two types of cells is similar — we’re assuming that’s the case until proven otherwise. And yes, I simplified the role of serotonin above. It’s associated with feelings of well-being when we’re talking about its role in the brain, but it has a variety of other roles elsewhere in the body.
And a couple more caveats: This study is small, with 49 subjects — 24 nurses from high-stress environments and 25 from relatively low-stress environments — partly because the investigators removed a lot of participants to control for smoking and alcohol consumption. Finally, the study relies on a bisulfite sequencing protocol to detect methylation, which means that inconsistent PCR amplification could have skewed the results. The team validated its findings by following up with an array-based method, but it’s important to note these things.
Still, that’s science. And without a few tentative steps steps forward, where would we be? Even more stressed, that’s where.
[I was stumped about how to portray both nurses and stress, or highly stressed nurses, so I opted for the pic above, Italian Nurses, which is by Flickr user jdlasica and is reproduced here under a Creative Commons license.]
Jukka S. Alasaari1, Markus Lagus1, Hanna M. Ollila1, Auli Toivola1, Mika Kivima ̈ki2,3, Jussi Vahtera2,3,, & Erkki Kronholm4, Mikko Ha ̈rma ̈2, Sampsa Puttonen2, Tiina Paunio1,5* (2012). Environmental Stress Affects DNA Methylation of a CpG Rich Promoter Region of Serotonin Transporter Gene in a Nurse Cohort PLoS One, 7 (9) DOI: 10.1371/journal.pone.0045813
Yu Bo is a highly respected, world famous chef who prepares modern versions of Chinese cuisine. His dishes are terrifically creative in their presentation of traditional Cheng Du providence flavors. Yu Bo’s process reveals the ‘essence’ of a dish, displaying it through modern culinary procedures and techniques.
This inventive process is similar to how biological information can be processed to reveal an essential disease profile. Breast cancer has many known associated single-nucleotide polymorphisms(SNPs). Most do not disrupt coding gene sequences. How can we discover which are causing disease?
Because exhaustive functional analysis of all GWAS results just doesn’t make sense, Richard Cowper-Sal-lari et al Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression (2012) Nature Genetics, propose the use of a new “integrative functional genomics” approach to explore relationships between breast cancer-associated SNPs and, as the authors phrase it, “…cistromes and epigenomes (that) lie at the source of cell identity”. An epigenome and a cistrome are genome-wide sets of epigenetic marks and transcription factor binding sites, for a specific cell type and cellular environment.
Integrative functional genomics works through a computational method called variant set enrichment (VSE) – followed by a second computational method termed Intragenomic replicates (IGR) to predict affinity of a binding motif based on a SNP sequence. The results are quite elegant. They show that the majority of risk-associated SNPs function to modulate the pioneer factor FOXA1, whose binding is necessary to alter the structure around genes to grant access for the transcription factor ESRI (estrogen receptor α).
My complements to the chefs!
Cowper-Sal Lari R, Zhang X, Wright JB, Bailey SD, Cole MD, Eeckhoute J, Moore JH, & Lupien M (2012). Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression. Nature genetics PMID: 23001124
It seems like every article about epigenetics in the popular press includes a sentence about how maybe, just maybe this new finding or other proves that Jean-Baptiste Lamarck was right some 200 years ago. He famously tied “acquired traits” — characteristics an individual accumulates during its life, such as muscular arms — into a broader theory of how species evolve.
The most recent version I’ve seen is in the Sept 8 New York Times opinion piece “Why Fathers Really Matter,” though it’s indirect and noncommittal, as Lamarck comparisons tend to be:
Epigenetics proves that we are the products of history, public as well as private, in parts of us that are so intimately ours that few people ever imagined that history could reach them. (One person who did imagine it is the French 18th-century naturalist Jean-Baptiste Lamarck, who believed that acquired traits could be inherited. Twentieth-century Darwinian genetics dismissed Lamarckism as laughable, but because of epigenetics, Lamarckism is staging a comeback.)
Does “staging a comeback” imply that Lamarck may have been right? Writer Judith Schulevitz leaves that as an exercise for the reader. (I mean, she’s careful not to make any concrete statements.) The rest of the article is a fairly interesting rundown of some interesting epigenetic effects — low calorie intake and heart disease rates, that kind of thing.
But about Lamarck: No, he was wrong.
Sure, you can have a higher risk of diabetes if your father ate a high-calorie diet before your were born, but that doesn’t mean that diabetes is adaptive to a high-food environment. Also, give it a couple generations, and without more of the same environmental stimuli, the epigenetic effects fade away. And there’s no evidence that epigenetic effects are additive in any way: could you use one epigenetic effect to increase your clan’s risk of diabetes generation after generation? Probably not, since there’s a limited amount of epigenetic modification, say DNA methylation, that a single gene — or set of genes — can accommodate.
alling epigenetics evidence for Lamarckian evolution is like mistaking a dimmer switch for an electronics technician. So it’s surprising to see that claim repeated so often, for example in this PLoS research article. And in the popular press here, here, and here. (I do like the bonus florid prose in that second one from The New Republic: “Epigenetic research suggests that organisms adapt much more quickly and in much more promiscuous conversation with their habitats than orthodox theories of natural selection allow for.”)
Still, let’s not forget what Lamarck got right. It’s true that the environment changes the physiology of organisms permanently over generations — just not directly, and not over just a couple generations. His evolutionary theory was apparently the first to lay out a coherent overall story that relied on natural laws, rather than miracles and magical thinking.
This step toward finding the natural mechanisms governing life played a major role in inspiring Charles Darwin and many more Enlightenment naturalists. Besides, the man named thousands of species, many of which still bear his name, like the blue jellyfish Cyanea lamarckii.
It’s a shame Lamarck isn’t appreciated for his real contributions, but that’s no reason to get all misty-eyed about his mistakes.
[Lamarck's statue in the picture above sits in Paris's Jardin des Plantes. The picture can be found here at Wikimedia Commons, and it's used on this page under a Creative Commons license.]
[Similarly, the blue jellyfish picture is from Wikimedia Commons, here. It's also used in this post under a Creative Commons license.]
Ou X, Zhang Y, Xu C, Lin X, Zang Q, Zhuang T, Jiang L, von Wettstein D, & Liu B (2012). Transgenerational Inheritance of Modified DNA Methylation Patterns and Enhanced Tolerance Induced by Heavy Metal Stress in Rice (Oryza sativa L.). PloS one, 7 (9) PMID: 22984395
So I was exploring the wide world of epigenetics research on the internet as we often do at E3, when I came upon this paper. T. Nguyen Duc et al. Nanobody-Based Chromatin Immunoprecipitation Methods Mol Biol. 2012;911:491-505. Now this ChIP protocol isolates a transcription factor from the lysate of the hyperther- moacidophilic archaeon, Sulfolobus solfataricus. Not as interesting for most of us as say, exploring the histone code in breast cancer cells. However what about this business of using of nanobodies rather than polyclonal antibodies for ChIP?
What are nanbodies (Nab)? These are single heavy chain only antibodies, produced by llamas or camels immunized with the antigen target. Their Vhh domain is subcloned so that nanobodies can be produced cheaply, by bacterial expression. See the explanation here at the web site for VIB, a life sciences research institute, based in Flanders, Belgium. The technique is patented. But VIB offers a service to produce nanobodies against your target. “In principle” nanobodies could replace monoclonal drugs such as Herceptin™, possibly obtaining better activity, with possible inhalation delivery to patients, and would be more economical to produce. I’m reading online that Nabs are 1/10 the size of regular antibodies and some are already in stage II clinical drug trials.
Beyond these clinical drug type goals, Nabs are being produced for lab applications as evident by the aforementioned ChIP paper and for the projects in Epigenetics labs here and here . I’m curious as to what researchers’ hands on experiences are with these nanobodies as tools for lab applications. Are they Nabs really as effective as polyclonals and cheaper than monoclonals? Have you seen impressive Westerns, in vivo cell, tissue or whole animal staining, or especially ChIP combined with any downstream DNA analysis?
Sometimes you don’t need Indiana Jones to solve an archaeological mystery, sometimes you need a geneticist.
The research article Matthias Meyer et al. A High-Coverage Genome Sequence from an Archaic Denisovan Individual. (2012) Science, proves that point. In the article the whole genome of a little girl who lived ~80,000 years ago, and belonged to an ancient human species called the Denosivans, is sequenced from a (big) tooth and a (tiny) knuckle. Listen here to an NPR interview on the subject. These results are why archaeologists have become as fired up as geneticists are, over next generation sequencing. NGS has recently had a tremendous impact on their field, by producing valid ancient DNA results from some incredibly rare and precious samples.
The mystery of human evolution remains to be fully described. One of the primary questions is, when did modern human beings diverge from European Neanderthals, the (apparently) larger, Denisovans from Eurasia, and the hobbit like, Homo floresiensis species from Indonesia? Of course we still can’t even be sure if there were other human types. Genetic results have already shown that we all share some of the genes related to speech & language – so these other human groups likely spoke. Why did our species alone survive? Here is a terrific overview of ancient DNA studies Rizzi, E. et al. Ancient DNA studies: new perspectives on old samples Genet Sel Evol. 2012; 44(1): 21. I also gathered elsewhere, that the DNA molecule is this phenomenally stable in bone due to the presence of the calcium based mineral, hydroxyapatite. Apparently genomic DNA wrapped in protein histones is also more abundant and stable, than mitochondrial DNA.
Intriguingly the next step in this sort of research could be based on epigenetics. Already ancient animal DNA methylation analysis has been demonstrated. See Llamas B. et al. High-resolution analysis of cytosine methylation in ancient DNA. (2012) PloS 7(1) The epigenome can provide insight on aging, disease, nutrition and the process of animal domestication. Scientists hope that epigenetics will demonstrate a role in rapid adaption to environmental climate change. Granted that the samples can be found, it would make sense that modern human’s unique adaption abilities will be reflected by the contrast in our epigenomes to those of our late, distant human relatives.
Or so it appears, based on research by Gunther Meinlschmidt and colleagues. When they exposed 76 people to a stressful simulated social situation, they found changes in the methylation of two genes within an hour. What’s more, those two genes—oxytocin receptor (OXTR) and brain-derived neurotrophic factor (BDNF)—are important to human behavior. The oxytocin receptor conveys the hormone oxytocin’s effect at several sites in the body, including the brain. BDNF supports existing neurons, encourages their growth, and functions in memory and learning.
The case isn’t perfectly conclusive yet, of course—with 76 subjects between 61 and 67 years old, the study could be larger. And the team measured gene methylation in blood samples—and not brain samples, of course—so it’s not clear that the same changes happen in our big thinking organs when we’re stressed.
Still, it’s a cool finding. The study’s big enough to identify real things, and it’s reasonable to suspect that blood and brain OXTR and BDNF genes get methylated under similar circumstances. Also, this study seems to come the closest to what the more, uh, optimistic and fanciful epigenetics fans have been claiming. Now, it definitely doesn’t show that gene regulation responds to intentional happy thought, but the mental state of psychosocial stress does seem to trigger methylation—and quick.
Gunther Meinlschmidt is director of the Research Department of Psychobiology, Psychosomatics, and Psychotherapy at Ruhr-University Bochum’s LWL University Hospital, and the rest of the team works from the University of Basel, the University of Trier, King’s College London, and Ruhr-University Bochum. I spoke with him the other day to ask him a few things about his research. Here’s what we talked about.
Is this the first study you know of that looks at the epigenetic effects of stress on specific genes?
As far as I know, yes.
It depends a little bit upon what you mean by stress. You’re probably aware of this early life-stress—looking at stress in terms of early adversity, how it has long-term effects on the epigenome. Most of these studies have looked at specific genes, most of the studies have looked at the GR [glucocorticoid receptor gene]. But some studies—there was a recent one in the Archives of General Psychiatry by scientists from Montreal—in which they look genome wide, so using chip technology.
But again this was looking at early adversity. So as far as we know, we are really the first to look at acute effects of psychosocial stress on specific genes.
Can you describe the stress test that the subjects go through?
We applied a stress test called the TSST, the Trier Social Stress Test. It’s perhaps the most applied test to induce psychosocial stress—so it’s been applied in hundreds of studies.
It consists of two phases. The participants are required to do mental arithmetic and to undergo a kind of mock job interview, so they have to present themselves [favorably]. What they do is relevant to them. They do this in a social situation, so there are two collaborators sitting in front of them with a camera and a microphone—it’s not real or working, but the two collaborators usually wear white clothes, and they’re instructed to be very neutral and strict. For example they ask the participants to start calculating again, if they make any mistakes.
The participants know, of course, that this is just for research. They know that this is not a real job interview. But the mere situation induces stress because of this individual personal relevance together with the social evaluative threat. It induces subjective changes in stress, but also a stress reaction in terms of activity of the HPA [hypothalamic-pituitary-adrenal] axis.
What was the most surprising result in your research?
One thing is that we didn’t know at all if you could see changes occurring so quickly after a psychosocial event. Of course, histones change very quickly [when triggered]. But in terms of DNA methylation, [there is a widespread impression] that these changes are very long-lasting changes, and that they respond rather slowly.
But we found changes occurring within an hour after a psychosocial stress [session] that itself lasted ten minutes. I think it really adds to the picture of DNA methylation as something that can respond more quickly—not as quickly as some of the histone modifications probably, but it’s rather quick. In the literature, there’s data in a Nature paper on cyclic changes in DNA methylation that regularly turn genes on and off. That showed that DNA methylation changed within—I think—one or two hours.
So it seems like we have to look at changes in DNA methylation following external triggers, like social events.
The genes that became methylated were the oxytocin receptor (OXTR) and brain-derived neurotrophic factor (BDNF). About the oxytocin receptor, what tissues is it most associated with?
I’d have to look at the literature to be perfectly accurate, but it depends. The oxytocin receptor is sensitive to estrogen, so it’s not just be a question of tissue. It also depends on [a person's] endocrine status.
Peripherally the oxytocin receptor is relevant when it comes to contracting smooth muscle, such as in the uterus, or to elicit milk ejection. For example in the uterus, the hormonal milieu can upregulate the receptor. And of course, there is the brain, where the oxytocin receptor is involved in a lot of behavioral effects. You’ll find it in different parts of the hypothalamus, such as the medial preoptic nucleus, for example.
You discovered methylation changes in oxytocin receptor in blood samples—can’t take brain samples, I guess.
Of course, brain is not possible. You could take biopsies [of other tissues], but it’s invasive and ethically problematic. It probably wouldn’t be possible to do this before and after stress. So very quickly it gets very complicated.
Scientists from Montreal did a couple of nice studies in post-mortem brains [Here's another. —Ed.], where you have really good target tissues. But of course, you can’t use this to address quick changes in methylation. They studied only long-term consequences of some early adversities by reconstructing them using databases of information from people who donated the brains.
How much does the state of oxytocin receptor in blood correspond to the situation in other tissues—brain and so forth?
That’s an important open question. There is data on the comparison of blood cells with mucosa from the gut, and there was quite a bit of correspondence in the epigenome across tissues.
But really, to say that what we picked up in the blood is related to the epigenetic status in specific brain areas—we really don’t know. That’s one question that we’d like to answer, for example, with animal models now.
What’s the next step in your research?
We picked up some of the genes that were interesting to us. We’re really moving in one of three directions. One is to assess epigenetic changes more globally, so not only specific genes, but gene networks. Or with chips, even more.
The other thing is to have a better understanding of the time frame of the changes, to have a more narrow description of the epigenetic changes.
And then a third question is to identify the mechanism. So: What drives these changes in DNA methylation? Are there some enzymes involved in the methylation machinery that are up- or down-regulated following psychosocial stress? And what are the factors that are changing them? Are they factors that we’re already aware of, such as the usual candidates, cortisol and other factors? Or are there further factors involved that really drive this methylation change?
[The picture above wasn't intended to be about stress, but I thought it fit pretty well anyway. It's by Flickr user sashafatcat, and it's used here under a Creative Commons license.]
Unternaehrer E, Luers P, Mill J, Dempster E, Meyer AH, Staehli S, Lieb R, Hellhammer DH, & Meinlschmidt G (2012). Dynamic changes in DNA methylation of stress-associated genes (OXTR, BDNF ) after acute psychosocial stress. Translational psychiatry, 2 PMID: 22892716
What was once surprising is now established. DNA methylation is not static after imprinting. All genes are not silenced “forever” through DNA methylation. But how does this dynamic and reversible mechanism work? As the hypothesis goes, 5hmC is a step in the de-methylation process initiated by Tet dioxygenases. The news today is that 5hmC is more than just a quick step, it has function.
In Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers. (2012) Nucleic Acids Research, 1-11. A.A. Serandour & S. Avner et al. show that conversion of 5mC to 5hmC activates enhancers – even pointing to it as an early step in the enhancer activation process. Can we all say functional signalling mark?
Some of you might be saying what are “enhancers” anyway? Enhancers are non-coding sequences which regulate long-distance gene expression. Their own activity is regulated by transcription factors. Enhancer sequences often vary significantly among different cell types. Although their sequences are not highly conserved, they can be ID’d through changes in the proteins that support DNA; histone marks, histone variant deposition and nucleosome stability.
For this study, samples of differentiating adipocyte cells and differentiating neural cells were run through a range of analyses, identifying new enhancers. hMeDIP-seq was used to select and analyze 5hmC genomic DNA. ChiP-seq was used to select for and analyze interactions among specific histone modifications, Tet1, and Meis1(a transcription factor). The FAIRE assay was used to demonstrate chromatin opening at enhancers. Transcriptome arrays were used to measure expression of differentiation genes. Newly ID’d enhancers were cloned into a luciferase reporter systems to establish their activities in vivo differentiating cells.
Data results were “heat maps of the relationships” at differentiation in the 2 cell line samples. I have to admit I’m terribly inadequate to explain the bioinformatics applied – see the paper for details. Simply put; When 5hmC is UP, hallmarks of ehancer activation H3K4me2 & H3K27ac are UP, Chromatin density is DOWN and differentiation associated gene expression levels are UP
Looks like 5hmC is an important signalling mark for initiating and maintaining cellular differentiation status, partially through regulation of enhancer activity. It makes sense since adult tissues and cells contain more 5hmC than both stem cells or most tumor tissues by immunolocalization. Cell specific hydroxymethylated enhancers are an influential layer of epigenetic instruction on DNA, attracting differentiation transcription factors and demethylating machinery like a magnet.
Sérandour AA, Avner S, Oger F, Bizot M, Percevault F, Lucchetti-Miganeh C, Palierne G, Gheeraert C, Barloy-Hubler F, Péron CL, Madigou T, Durand E, Froguel P, Staels B, Lefebvre P, Métivier R, Eeckhoute J, & Salbert G (2012). Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers. Nucleic acids research PMID: 22730288
Typical of epigenome scans, this one doesn’t make any clear links between methylation states and any diseases, though the researchers make a couple plausible connections, for example, suggesting that demethylation affects the AHRR gene’s role in fibroblast apoptosis in lungs. In any case, the data will be very useful to epigeneticists in general.
Researchers from the NIH National Institute of Environmental Health Sciences, the Norwegian Institute of Public Health, the Haukeland University Hospital in Bergen, Norway, Duke University, and several other institutions published the paper online at the NIEHS website under the title “450K Epigenome-Wide Scan Identifies Differential DNA Methylation in Newborns Related to Maternal Smoking During Pregnancy.”
In one approach that avoids some of the ambiguities of surveys, the researchers use continine levels in the blood of pregnant moms to ascertain smoking behavior in the main subject group, which they drew from the Norwegian Mother and Child Cohort Study. But they used self reports to determine smoking behavior in the 36 people who were part of the replication study, and drew these subjects from the Newborn Epigenetics STudy, known as NEST.
The investigators identified 26 CpGs as significantly changed, when comparing non-smoking moms to smokers, and they were mostly associated with four genes, which they narrowed to three. Comparing these CpGs to the replication study’s epigenomes — of smokers and non-smokers — the team verified its choices.
- Growth factor independent 1 transcription repressor (GFI1)
- Aryl-hydrocarbon receptor repressor (AHRR)
- Cytochrome P450 isoform CYP1A1
Both AHRR and CYP1A1 are involved in detoxification, and a previous epigenome scan of smokers and non-smokers also flagged AHRR. So, this study seems to show for the first time that this response occurs in fetuses.
GFI1 is mostly known as a development gene — it’s involved with the inner ear, for example, and it influences apoptosis, differentiation, and more. It appears to do this at least partly through roles in histone modification and RNA splicing.
So, the authors suggest, this gene at least has the potential to be broadly influential in the development of a human being, possibly enough to explain some health effects.
[The ashtray pic above is by Flicker user cipher, and it's used here under a CreativeCommons license.]
Joubert BR, Håberg SE, Nilsen RM, Wang X, Vollset SE, Murphy SK, Huang Z, Hoyo C, Midttun O, Cupul-Uicab LA, Ueland PM, Wu MC, Nystad W, Bell DA, Peddada SD, & London SJ (2012). 450K Epigenome-Wide Scan Identifies Differential DNA Methylation in Newborns Related to Maternal Smoking During Pregnancy. Environmental health perspectives PMID: 22851337
The World Anti-Doping Agency (WADA) has committed $50 million US dollars to research since 2001 (see their grant applications & projects here). Since 2007, some of those research funds have gone to the emerging problem of gene doping. Read about the first public evidence of gene doping, from the trial of the German track coach Thomas Springstein., in the NYT article Outlaw DNA.
So what is gene doping? WADA defines it as “the transfer of nucleic acids or nucleic acid sequences’ and/or ‘the use of normal or genetically modified cells with the intention to enhance sports performance.” Gene doping is based on a vector containing a therapeutic gene coupled with a regulatory element, delivered to somatic cells either in, or ex vivo. Plasmid based vectors have poor transfection efficiency and short duration of expression. Viral based vectors can cause deadly immune responses to the vector, but are difficult to detect by using anti-virus antibodies, given that they are based on commonly contagious, wild type viruses. Both gene doping methods can lead to autoimmune responses to the endogenous gene product made by the human body. Yikes, right?! There is no safe gene doping. The attraction lies in the idea that gene doping has been thought of as “undetectable”. However unlikely, gene doping is still an alternative to the sophisticated drug doping protocols, estimated to be used by up to 10% of high level athletes to stay just below detection thresholds.
Repoxygen™ is an example of technology at risk for gene doping, featured in the review by Elmo W.I Neuberger et al., Detection of EPO gene doping in blood. (2012) in Drug Testing and Analysis. Endogenous erythropoietin (EPO) circulates in our blood, stimulating the differentiation of red blood cell precursors cells in bone marrow. Thereby increasing our oxygen delivery. In Repoxygen™, a human EPO gene is delivered by intramuscular injection, within a viral vector with self regulated expression through a hypoxia response element. Many EPO-like or EPO stimulating drugs are available for treating anemia. Promising results in mice studies of Repoxygen™ were not enough for the developer, Oxford Biomedica to follow up with clinical trials in humans because of the presence of these alternative EPO drugs. Therapeutically these other drugs used by people with heriditary anemia, IDS, Cancer, etc require multiple injections. So technology like Repoxygen™ is still relevant. Several studies have used plasmic ‘mini circle’ vectors to improve safety of the technique in primates.
Currently, WADA prescribes the indirect Athlete Biological Passport (ABP) system to monitor athletes for responses due to doping over time. Indirect measures should catch any doping, no matter the method in theory. Again, this isn’t really ideal since cheaters can use doping protocols to beat this system. Some projects funded by WADA aim to develop detection methods specific to gene doping. A current focus of this research uses transcriptomics. Specifically, highly stable, circulating microRNA from serum, is being used to look for gene doping biomarkers. This approach will certainly require a large sample of the population and good bioinformatics to get it right. It may all work out. It’s conceivable that athletes will be seeking personalised genomics in the future to assess their performance abilities based on their gene expression. You can’t stop progress.
Neuberger EW, Jurkiewicz M, Moser DA, & Simon P (2012). Detection of EPO gene doping in blood. Drug testing and analysis PMID: 22508654