- prof premraj pushpakaran on New model for Down’s Syndrome Developed Using X Chromosome Inactivation
- Emmy Han on Addiction and our Epigenome
- Harvesting Plant Methylomes using MspJI-seq | Epigenetics Experts Blog | Roberts Lab on Harvesting Plant Methylomes using MspJI-seq
- 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?
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It’s my birthday. I’m of a certain age. My methylome is diminishing…and I’m taking it personally. What can epigenetics tell me about halting this process?!
There is some context to ageing. As we age, our global methylation levels decrease. Yet, gene promoter methylation increases, especially for genes involved in regulation of developmental patterns. Ageing is like a slow version of carcingogenesis. The biomarker patterns differ between our tissue types. But humans generally share these systemic ageing epigenetic signatures. This fact is the basis for techniques used by forensic scientists to discriminate the age of a criminal suspect, on DNA evidence.
On the other hand, there is “epigenetic drift”. That is the individual variability among people in their personal functional decline and disease vulnerability. We see that epimutations occur as epigenetic mechanisms relax. For example, DNMT expression and activities change. DNMT1 and 2 are decreased, while DNMT3a increases.
We just don’t know exactly how all this happens, or how to stop it. BUMMER!
I’ve read about the lifestyles of super-centenarians – looking for tips. They all have different lifestyle claims for their longevity. Some have a lot of kids some don’t. Some smoke for 40 years. Some drink everyday. The only pattern among them that I detect, is their sustained youthful appearance and abilities over time. This latest epigenetics ageing review paper on diet, states that “ part of ageing is due to an epigenetic program set during development.”
It seems like we are all born with a countdown setting that can be influenced only incrementally. But you can bet your life it’s still worth trying!!!!
In Cortijo, S. et al. Mapping the Epigenetic Basis of Complex Traits (2014) Science
Express. Researchers from the University of Groningen Bioinformatics center, and their collaborators, have made the exciting discovery that epigenotypes in plants result in complex phenotypes that are stably inherited, and subject to selection.
The team prepared inbred Arabidopsis samples with the same genomes, but differing methylomes. Methyl-seq analysis showed that certain differentially methylated regions (DMRs) behave as quantitative trait loci (QTL epi). These QTL epi were demonstrated to be responsible for 60-90% of two complex traits, flowering time and primary root length. The same QTL epi were also found functioning in wildtype Arabidopsis.
Co-Team leader, Assistant professor Frank Johannes explains ‘We used the same method to locate regions in the DNA, not with different sequences but with different epigenetic marks that contribute to certain traits in the plant. This is a breakthrough, because it changes the way we view genetics. And it may even be of huge economic importance.’
From a plant evolution perspective, this new information could answer the controversial “problem of missing heritability”.
Phenotypes are traits expressed by organisms in a specific environment. When you think of your own ethnicity, you might mostly be considering your appearance. These visible characteristics are associated with where your ancestors came from, of course. Now, don’t take this personally. But, biomedical researchers associate your ethnicity with risks. Why?! Because ethnicity can be associated with phenotypes with health risks.
We once expected that by sequencing the entire human genome, the basis of human diversity would be revealed. After all, your traits are based on your genes, right? Unfortunately, it has been difficult to demonstrate how our genomes produce the varieties of human phenotypes. Case in point, most human mutations / gene variants are located in non-coding regions of our DNA. Research focus has shifted to look behind the “genome sequence” curtain, so to speak. Behind the curtain, there is a complicated system of inter-connected epigenetic machinery, modulating gene expression. Human genome databases are the basis for this exploration.
How does epigenetics relate with phenotypes? For the paper, Holger Heyn et al. DNA Methylation contributes to natural human variation (2013) Genome Research, the authors interrogated the methylomes of three ethnic groups using methylation sequencing arrays. They report several fascinating findings that struck me.
- Their method of GWAS targeting methylation clusters blood & tissue samples by ethnicity.
- Methylation appears to act as an evolutionary established mediator, within the relationship between genotype and epitype.
- Local selective pressure may induce independent epigenetic variants.
- “Pop-CpGs”* could aid genetic variance GWAS. (*definition below)
In this study, three ethnic groups were sorted by methyomes alone. The researchers started with 288 samples B-lymphoblastoid cell lines, produced by EBV immortalization. B-lymphoblastoid cell line genomic DNA samples were sourced evenly from Caucasian-American, African-American and Han Chinese American people. (BTW Fun fact: Han Chinese are the largest ethnic group in the world! )Methylation profiles were assessed using Illumina’s DNA methylation BeadChip platform. This array covers 96% of CpG islands, totaling 485,000 genes, at single nucleotide resolution. Libraries of genomic DNA are bisulfite treated. This process converts un-methylated cytosines to uracil. Samples are each exposed to methylated bead probes, and un-methylated bead probes. A matched DNA probe causes single-base extension of that sample sequence, incorporating a labeled ddNTP, that is then detected through fluorescence staining. By this sequencing array method, the group found 1373 differentially methylated CpG sites that clustered primarily by donor ancestral geographical origin. Termed by the authros as , “Pop-CpGs”, these are cytosine phosphodiester bond guanine sites that have population specific differential methylation. Of the 1373 sites, 439 pop-CpG sites for ethnicity were also shared in primary blood samples. They also present results with regard to human tissues, Hepatitis B infection risk and xenobiotic response factors.
The researchers also discovered evidence for other types of gene expression regulation. Their analysis of pop-CpGs showed an enrichment with two histone enhancer marks and a histone heterochromatin mark. … transcription factor binding sites. They performed expression studies to show the DNA methylation at some of these pop-CpGs was associated with gene repression and activation. Some pop-CpGs are located in those non-coding areas. Therefore it appears that regulatory elements other than promoters, are tapped for human variation.
Their results are a small survey of how the human epigenetic system works with our genetic system. A third of pop-CpG were not directly associated with any underlying genetic variation. Two thirds were. So, most of the time the DNA sequence sets up the context for methylation. Ethnicity in these study groups, is linked to differences in drug metabolism, response to external stimuli, sensory perception and disease risk. The authors’ suggestion that independent pop-CpGs could be the result of things like diet, pathogen, drugs, pesticides, carcinogens and hormone exposures makes sense. More GWAS studies using Pop-CpGs should lend support the epitype induction idea.
Heyn H, Moran S, Hernando-Herraez I, Sayols S, Gomez A, Sandoval J, Monk D, Hata K, Marques-Bonet T, Wang L, & Esteller M (2013). DNA methylation contributes to natural human variation. Genome research, 23 (9), 1363-72 PMID: 23908385
Recently I had the occasion to take a trip to Manhattan, and visit at the Dream Downtown hotel. The designers of this hotel put a great deal of effort into creating an unusual, cutting edge modern environment, worthy of its name. You enter from the street, into this darkened…yet vast lobby. Two stories above your head, shimmery waves of day light shine through a glass bottomed, swimming pool. In the restaurant, a dramatic field of golden glass globe lights spray across the ceiling. There is an actual rowboat “floating” on a vertical koi pond wall. The hotel elevators have four fully mirrored walls. Walking around this hotel pushes you to approach spaces that suggest impossibility. You leave your comfort zone as you enter. Is this really what I am seeing? Is this safe? In the end you are rewarded with a calming experience. Calming, because your human mind has to be “present” to adjust to the challenging sensory environment.
Pushing RNAi studies towards clinical medicines has required a similar fortitude by researchers. There is a new focus on the cellular RNA environment. How extensive is the influence of RNA systems in organisms? Can RNAi be used to control gene expression precisely enough in people? Not to mention, do I have the skills to work with RNA in the lab?
RNAi is a system used by eukaryotic cells to regulate gene expression, and to defend against viral infections. While, RNAi technologies use RNAi cellular machinery to produce lab designed small interfering RNA (siRNA) to target and knock down gene expression. First, sequence coding antisense RNA is cloned into a plasmid or viral vector. There are two types of vector systems. One has Short Hairpin RNA, with both sense and anti-sense expressed by one promoter. The other is a tandem system with sense and anti-sense expressed by two separate promoters. Transfection into cells can be carried out by either electroporation or lipid formulations. Vector expressed siRNA couples with cellular RNA-Induced Silencing Complex (RISC) capable of degrading messenger RNA. The siRNA molecule recognizes and binds to its targeted cellular mRNA, producing the “red flag” dsRNA molecules. One siRNA molecule works in this fashion to eliminate multiple copies of an mRNA sequence.
A clinical goal has been to use RNAi technologies as gene therapy based medicines. For example to “knockdown” oncogene proteins, viral proteins, or mutated proteins causing diseases. Advantages and disadvantages have been discovered. Viral vector delivery systems carry the risk of causing tumors. Lipid nanotechnology is an improved, inert delivery system. However, transfection is still transient, requiring maintenance. Also, siRNA is designed to be no longer than 21-23 bp, to avoid triggering an animal host interferon system attack. “Off-target silencing” can be a problem in organisms, too. Still, there are numerous ongoing phase I and phase II clinical trials looking overcome such challenges.
Encouraging Phase I clinical trial results were reported in the New England Journal of Medicine paper, Safety and Efficacy of RNAi Therapy for Transthyretin Amyloidosis. Lipid-based nanotechnology delivered RNAi was used to knock down of mutated (and non-mutated) forms of the protein Transthyretin (TTR). Transfection was targeted to the liver, where TTR is produced. TTR functions to transport vitamin A and hormones throughout the body. In various types of Transthyretin Amyloidosis. mutated TTR forms plaques in different areas, (i.e. peripheral nervous system, autonomic nervous system, heart, kidneys, eyes, and the gastrointestinal tract). The disease is progressive and life threatening. The only current treatment is a liver transplant. Lipid-based delivery RNAi appears to be a good match for the liver. The chances of off target silencing are certainly low. The liver breaks down lipids to produce most of the body’s cholesterol. The liver is a uniquely regenerative organ, as well. In live donor transplants, the donor and recipient start out with half a liver. Six weeks later each has a full sized regrown liver. (Save that fact for a party.) In this study, their lipid nanoparticles were safe and their best formulation results had a 70% reduction at day 28 in monkeys, 90%+ reduction after 5 doses. Knockdown of TTR in human patients observed at one dosage were identical to the primates. A caveat is that long term treatments could result in vitamin A deficiency, but the authors predicted that other independent mechanisms of vitamin A transport could compensate.
Who dares nothing, need hope for nothing.
Coelho T, Adams D, Silva A, Lozeron P, Hawkins PN, Mant T, Perez J, Chiesa J, Warrington S, Tranter E, Munisamy M, Falzone R, Harrop J, Cehelsky J, Bettencourt BR, Geissler M, Butler JS, Sehgal A, Meyers RE, Chen Q, Borland T, Hutabarat RM, Clausen VA, Alvarez R, Fitzgerald K, Gamba-Vitalo C, Nochur SV, Vaishnaw AK, Sah DW, Gollob JA, & Suhr OB (2013). Safety and efficacy of RNAi therapy for transthyretin amyloidosis. The New England journal of medicine, 369 (9), 819-29 PMID: 23984729
We are all addicts in some sense. We each take some actions repeatedly, despite their perceived negative consequences. My favorite comedian is Jim Gaffigan. In my view, Gaffigan’s famous bit on McDonalds, sums this idea up.
“We all have our McDonalds. Has your mother ever made anything as good as a McDonalds fry?…NOT EVEN CLOSE. I’m tired of people thinking they’re better than McDonalds. But really you have your own McDonalds. Maybe instead of buying a Big Mac™, you read US weekly. Hey that’s still McDonalds. Maybe your McDonalds is telling yourself that Starbuck Frappichino is not a milk shake. Or maybe you watch Glee. Its ALL McDonalds. McDonalds of the soul. Momentary pleasure, followed by incredible guilt, eventually leading to cancer. “
So called “behavioral addiction” is just part of our human nature.
The dire straits of cocaine abuse
Unfortunately being prone to drug addictions is also very human. Cocaine abuse is especially monstrous. Taking cocaine releases the pleasure neurotransmitters dopamine, norepinephrine, and serotonin. At first the effect is bliss. Soon after, cocaine disrupts arousal, fight or flight, appetite, mood and sleep systems in the brain. All pretty miserable. Repeated use causes the gradual depletion of the neurotransmitters and dis-regulation of all these systems. The bottomless pit of addiction to this drug is profound. Lab primates with unlimited access, will take cocaine until they die with seizures. One study demonstrated that primates will push a bar 12,800 times for a single dose!
Cocaine addiction disorder is a plague to society, with associated misfortunes comparable to, or surpassing any disease. Addiction leads to numerous psychiatric and cardiovascular problems. In the USA population, almost 1/3 of homicide victims are associated with cocaine use. As are about 1/5 of suicides. Understanding the biology of addiction in order to develop treatments is critical to our health as a society. Unlike primates, our human tendency for addiction is tempered with our ability to plan our actions based on risk assessments.
Epigenetics of transgenerational inheritance
Fair M. Vassoler, Ghazaleh Sadri-Vakili Mechanisms of transgenerational inheritance of addictive-like behaviors (July 2013) Neuroscience. Explores the possible modes of how drug abuse changes the epigenome of future generations. They also present some preliminary results of their new model in rats.
The authors review relevant research results. They note examples demonstrating three modes of epigenetic mechanisms of transgenerational epigenetic inheritance. These generally include DNA methylation, post-translational histone modifications and small non-coding RNAs. They discuss other environmental stimuli shown to produce transgenerational inheritance. These include examples of behavioral or social transmission to offspring, environmental toxins, modes of stress and diet. The authors point out that these various environmental inputs likely share the same underlying epigenetic processes leading to adaption.
The group developed a model of paternal cocaine abuse in rats. Male rats were trained to self administer cocaine, and were allowed to do so for the 60 day duration of spermagenesis. Then they were mated. The behavioral results were that “…male, but not female, cocaine-sired offspring, demonstrate delayed acquisition and decreased intake of cocaine during adulthood … “ For people in the real world, a family history of drug addiction increases your susceptibility. However the opposite held true in their experiments so far. I repeat, the first generation offspring (F1) actually show increased resistance to cocaine.
The scientists had previously reported that cocaine-induced increases in brain-derived neurotrophic factor (BDNF) were associated with increases in acetylated histone H3 (H3K9K14ac2) in the medial prefrontal cortex (mPFC ). In this study they found that BDNF mRNA and protein from the mPFC increased, due to increased acetylated histone H3 (H3K9K14ac2). But only in male offspring. I wonder why that is? The next test will be what happens in the next generation (F2) , the “grand-rats”, so to speak. That’s important because that generation are not produced from the gametes of the first drug addicted generation.
These ideas are all kind of wild, since we didn’t even know until recently that traits could be inherited via the epigenome. We thought the imprinting process totally cleared the genome. But instead there is an impression left from these key experiences in the previous generation. Further research will aim to work out the mechanisms behind drug resistance….and perhaps all transgenerational epigenetic adaption.
Vassoler FM, & Sadri-Vakili G (2013). Mechanisms of transgenerational inheritance of addictive-like behaviors. Neuroscience PMID: 23920159
I have a special cousin who has Down’s syndrome. He’s cheerful. He’s kind. He’s hardworking, He has a photo collage of all his extended family in his room. We all love him dearly. I admire my aunt who dutifully raised him, with the same discipline as her other healthy son. Unfortunately, Down’s syndrome produces an additional challenge, as my cousin ages…memory loss from Alzheimer’s disease.
That is why I am so grateful to researchers like Jeanne Lawrence of Umass Medical school in Worcester Massachusetts. Last week her group published this impressive article Jun Jiang et al. Nature Translating dosage compensation to trisomy 21. (2013) Nature. Their notable comment was “Our hope is that for individuals and families living with Down’s syndrome, the proof-of-principle demonstrated here initiates multiple new avenues of translational relevance for the 50 years of advances in basic X-chromosome biology.”
To understand this paper, you need to understand dosage compensation. This epigenetic regulatory system prevents twice the expression of all the X chromosome genes in females. It works like this. The gene XIST expresses non-coding RNA that randomly targets one of the X chromosomes in each cell during blastocytosis. The silence is stabilized by hypermethylation of promoter CpG islands. The chromosome is condensed, forming what is termed a “barr body”. Interestingly, around 10% of the genes in the barr body somehow escape inactivation. It has been known for some time that inactivation spreads linearly along the X chromosome. We also knew that a non-sex chromosome can be attached in the lab to an X chromosome undergoing inactivation, so that it too will be silenced. The inactivated X chromosome or Barr body is only reactivated as an X chromosome during oocyte formation. That way both sets of the potential grandparents’ X chromosome can be available for inheritance. In cases of women with Triple X, or three X chromosomes two of the three X chromosomes will be silenced. So there must be some feedback mechanism protecting one.
The basic cause of Down’s syndrome is the extra copy of chromosome 21. The researchers asked, “Would the XIST mechanism work on that extra copy?” The answer was yes.
This cell culture model was designed thoughtfully to be useful in basic research as ‘trisomy correction in a dish’. Inducible doxycycline-controlled XIST transgene was inserted into chromosome 21, of Down’s syndrome iPS cells, via zinc finger nuclease (ZFN)-driven targeted addition. One to all three copies of chr 21 could take in XIST. Impressive enough, since such a large gene has not been inserted by this method before. Heterochromatic epigenetic marks, such as histone post translational modifications, were used to show whole chromosome silencing similar to barr bodies. They also showed 100% silencing of various individual chromosome 21 genes, i.e. RNA FISH. Including the gene that encodes β-amyloid precursor protein, that is over expressed in Down’s characteristc Alzheimer’s.
However in any of their cultured clonal lines, they observed that other epigenetic adaption processes work to silence the inserted XIST genes over time. Or otherwise the XIST gene is just lost by cells. XIST repression had an additional “defined effect on the genomic expression profile, and reverses deficits in cell proliferation and neural progenitors, which has implications for hypocellularity in the Down’s syndrome brain”. Cells likely have measures in place to ensure correct overall dosage from a pair of non-sex chromosomes.
Since X chromosome dosage is so stable, I wonder what other mechanisms are at work? What do iPS cells in this model have in common with cells undergoing oocyte formation?
Excitingly, the authors point out that their findings are a boost to future translational research and gene therapy research. Parallel cultures can be compared for differences in genome-wide pathways and Down’s syndrome related cell pathologies. The model iPS cells can be differentiated, i.e. neurons for studies. Hopefully someday therapies could be even be developed for Down’s adults, or could treat Down’s syndrome in utero. I’m just going to say it. The ideas from these researchers are mind blowing.
Great news especially for plant labs! Researchers from BGI, and their collaborators, have shown that using the restriction enzyme MspJI upstream of next generation sequencing, is very effective to produce high resolution, ~50% representative plant methylomes. MspJI-seq Arabidopsis methylome data was BETTER than MeDIP-seq and MBD-seq data. MspJI-seq data sensitivity and specificity was comparable with whole genome bisulfite sequencing, (WGBS) data. Geneticists have used Arabidopsis, as a model organism, to produce much of the basic knowledge concerning DNA methylation in plants. However, most plants generally have very large and repetitive genomes – compared to mammalian cells. That translates into very large price tags for bisulfite sequencing. (Yikes!) The BGI authors stated that they have produced a plant methylome mapping method that could “be feasible, reliable, and economical in methylation investigation.”
So what do botanists know about plant DNA methylation? Cytosine methylation in plants occurs at CpG, CHG and CHH sequences. “H” standing for A,C or T. DNA methylation has been shown to have critical roles in plant environmental response and development. Methylomes can affect plants across generations. Methylation also functions to protect plant genomes from transposons which can reek havoc on plant chromosomes.
MspJI-seq is an avenue for further plant methylome research. More work is needed to show how all of these downstream effects are produced exactly. As well as what mechanisms control plant DNA de-methylation. I’d love to see the MspJI-seq method applied to crop plants, i.e. rice, maize. Also, I read that some epigenetic processes are shared with mammals. It can be easier to use plants as models to study the impact of these processes in human diseases.
Please have a look at this paper to learn the method details. Huang X, Lu H, Wang JW, Xu L, Liu S, Sun J, Wang J, Gao F. High-throughput sequencing of methylated cytosine enriched by modifcation-dependent restriction endonuclease Msp JI. (2013) BMC Genet.
The recent news about Angelina Jolie getting a prophylactic double mastectomy is both sad and encouraging. Women, and their physicians, are becoming more aware of individual breast cancer risk. They are willing to use any available treatments to reduce that risk, and promote health.
Tamoxifen is a breast cancer drug success story. It works by competing with estradiol for estrogen receptor protein. Thereby inhibiting the Erα (estrogen receptor). See http://www.drugs.com/pro/tamoxifen.html Tamoxifen 1st significantly improves survival. 2nd reduces recurrence. 3rd reduces the incidence of breast cancer in high risk women. The drawback is that the tumors need to be overexpressing estrogen receptor, or ER+. Cancer that is “estrogen receptor negative”, or ER- now has a comparably worse prognosis. This is because hormone therapy using Tamoxifen or other selective estrogen receptor modulators (SERMs) doesn’t work for ER- cells.
An intriguing idea for treatment for ER- breast cancer was explored in the PloS ONE paper, Bioactive Dietary Supplements Reactivate ER Expression in ER-Negative Breast Cancer Cells by Active Chromatin Modifications. There are many DNMT and HDAC inhibitor synthetic drugs actively being researched. The challenge has been their low specificity and high cytotoxicity. The natural compounds green tea polyphenols (GTPs) and sulforaphane (SFN) target both DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). As per the title, the authors showed how GTPs and SFN work synergystically to reactivate the estrogen receptor in ER- cancer cell lines. The right dosage combination made the cancer cells susceptible to Tamoxifen treatment. They examined the underlying molecular mechanism.
Investigating natural anti-epigenetic compounds is cutting edge cancer research. Ideally, combinational treatment of hormone therapy with diet supplements will prevent cancer, or halt its progression. The right treatment will reliably reduce an individual patient’s risk. And an ounce of prevention is worth a pound of cure!
Here’s another great advance in methylome sequencing. You all know about bisulfite sequencing, the “gold standard” method. Unfortunately it’s expensive. It also requires a lot of sample, due to DNA degradation. There are enrichment methods, like MeDIP-seq, that are relatively cheap. However, there is the drawback of CpG density bias. Excitingly, there is a new enzyme based enrichment method, called TamC-Seq that requires less sample, less money, and provides excellent coverage for genome-wide profiling. The devlopers are from the He group, University of Chicago. The paper is Liang Zhang et al. Tet-mediated covlent labelling of 5-methylctosine for its genome-wide detection and sequencing. (2013) Nature Communications, (4) 1517
So how does it work? Their protocol uses mouse Tet (Ten-eleven translocation)-1, (or mTet1) enzyme expressed in a baculovirus system. First 5hmC is protected with a glucose using Beta-GT. Next, in a “One pot” procedure, the mTet1 converts 5hmC from 5mC, which is then immediately glycoslated by Beta-GT with a modified glucose moiety(6-N3-glucose) . Then all the original 5mC is labeled with biotin via click chemistry. Time for affinity biotin/streptavidin purification, followed by sequencing.
The researchers used mouse embryotic stem cells, as well as human breast cancer cell lines, to compare methylome data with other methods. TamC-Seq was efficient at providing more methylation site coverage than MeDIP-Seq. TamC-Seq captured a wider range of CpGs, showing less density bias than MeDIP-Seq. The TamC-Seq data was concordant with Bisulfite-Seq data.
The same research group is also working on RNA epigenetics. Interesting! Check out their web site.
Zhang L, Szulwach KE, Hon GC, Song CX, Park B, Yu M, Lu X, Dai Q, Wang X, Street CR, Tan H, Min JH, Ren B, Jin P, & He C (2013). Tet-mediated covalent labelling of 5-methylcytosine for its genome-wide detection and sequencing. Nature communications, 4 PMID: 23443545
New England Biolabs is well known for its extensive in house research programs – churning out numerous publications every year. The role of hydroxymethylation as a possible cancer biomarker is a topic of keen interest for all Epigenetics researchers. So, NEB researchers are especially enthused about their recent publication in Cell, along with their collaborators from Emory University School of Medicine.
Sun, Z. et al. High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine in Mouse Embryonic Stem Cells. (2013) Cell Reports 3, 567-576. describes the Aba-seq method, an AbaSI enzyme based high-resolution hydroxymethylome mapping. (Open access.)
In nature, AbaSI is a weapon in the arms race between bacteria and bacteriophages. Wildtype bacteriophages such as T4, are resistant to most restriction enzymes due to their 5-hmC (hydroxymethylated) and 5ghmC (glucosylhydroxymethylated) DNA. However this type of bacteriophage DNA “armor” is no match for AbaSI, or other members of a family of restriction enzymes produced by bacteria, to stop T4 replication.
NEB scientists had isolated, prepared and characterized AbaSI to show ~10,000 fold higher specificity between 5ghmC and 5-mC. This feature brought the teams closer to the goal of developing a method to explore the mammalian hydroxymethome at near single base resolution. See figure 1B for the Aba-seq method overview.
For epigenetics research, the Aba-seq method has advantages over other methods developed to analyze the hydroxymethlome at high resolution sequencing. Keep in mind, only a a tiny fraction of mammalian DNA is hydroxymethylated (~0.1%). This method is not harsh on DNA samples. Only a small amount of DNA is needed 100ng. 5HmC sites with low occupancy can be reliably detected. Also, data analysis is straightforward.
The 5hmC pattern found in mouse embryonic stem cells by the researchers suggests a dependence on TET enzymes accessibility in some areas, and a demethylation intermediate or poised epigenetic state in others. The ease of this enzymatic method certainly contrasts to the complicated nature of epigenomes!
Sun Z, Terragni J, Borgaro JG, Liu Y, Yu L, Guan S, Wang H, Sun D, Cheng X, Zhu Z, Pradhan S, & Zheng Y (2013). High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells. Cell reports, 3 (2), 567-76 PMID: 23352666