- 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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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
Recent surprising evidence has shown that metastatic tumors usually do not vary in their genomes within an individual. Yet, these tumors behave differently at different sites around the body. Does that mean that epigenetic profiling will be too variable to target for cancer treatment? In a word, no.
Martin J. Aryee et al., from Johns Hopkins, have published their work in DNA Methylation Alterations Exhibit Intraindividual Stability and Interindividual Heterogeneity in Prostate Cancer Metastases in Science Translational Medicine. They looked at methylation signatures, including total methylation and allele-specific methylation (ASM) in lethal metastatic prostate cancer, among tumors from 24 donors. Methylated DNA was enriched from the genomic DNA using a Methyl-CpG Binding Domain (MBD) -based capture. Their MBD-SNP assay provided total methylation, ASM and copy number concurrently for each sample. Microarray analysis and computational analysis were used downstream to produce “cityscapes” of visualized data.
The epigenetic and genetic data varied a lot among the individual donors. Yet intriguingly, static epigenetic profiles were seen among metastatic tumors for each individual donor. This means that although metastatic prostate cancer tumors are somehow able to overcome treatments like the removal of androgens (surgical or medical castration), there are still “non-moving” epigenetic biomarkers targets to aim at to develop new treatments. The authors point out that it will be important to distinguish between the driver and passenger DNA methylation alterations in carcinogenesis. So there’s all that to still sort through in functional studies to determine targets with casuality in the disease progression. The stable methylomes within individuals supports the goals of personalized medicine in prostate cancer treatment – so it’s definitely worth the effort.
Aryee MJ, Liu W, Engelmann JC, Nuhn P, Gurel M, Haffner MC, Esopi D, Irizarry RA, Getzenberg RH, Nelson WG, Luo J, Xu J, Isaacs WB, Bova GS, & Yegnasubramanian S (2013). DNA methylation alterations exhibit intraindividual stability and interindividual heterogeneity in prostate cancer metastases. Science translational medicine, 5 (169) PMID: 23345608
Finally there’s research comparing the epigenetic marks of human brain neurons to those of other primates, and it’s found real differences that make us function in a unique way. Do these epigenetic modifications help give us the brainpower for reflection, sentience, sapience, consciousness, and so forth? I’m not a gambler, but since primate neuron-specific genes don’t show a whole lot of difference from one another in their protein-coding sequences, that’s where I’d put my money. If I really had to.
With only one study to look at so far, this line of inquiry is in its infancy, to be sure. No one else has looked at the epigenetic component of human brain evolution. Hennady Shulha, Jessica Crisci, and Schahram Akbarian at the University of Massachusetts Medical School — and colleagues — took that first step with research they published in PLoS Biology late last month.
[Update 12/20/2012: Twitter friend @ed vautier points me to this study in Epigenetics, which documents the evolution of additional CpGs in humans, compared to non-human primates, and looks at their relative methylation levels. It's not neuron-specific, but it's in the same bucket as this study, so I thought I'd note it here. And so I have. And there you have it.]
What they find is this: Human prefrontal cortex neurons sport 33 epigenetic modifications — histone H3 trimethylated at lysine 4, or H3K4me3 — in genomic locations where macaques and chimps are much less likely to feature them. These H3K4me3 marks tended to appear near each other, and when the research team used chromosome conformation capture to look more closely at the region around one gene, DPP10, they discovered that chromosome looping appears to allow two nearby H3K4me3 modifications to come into contact.
But although H3K4me3 marks often lead to higher gene expression levels, this interaction seems to cause down-regulation of DPP10 through an interesting mechanism. As it turns out, increased anti-sense DPP10 RNA might be the answer. When the team did RNA-seq and quantitative RT-PCR on cortex neuron samples from separate human subjects, they found high levels of DPP10‘s anti-sense RNA and lower levels of DPP10 sense mRNA, as compared to the analogous macaque and chimp neurons.
The team also found the anti-sense RNA at higher levels in the human subjects’ neuron-rich prefrontal cortex layers II-IV, but not in neuron-poor layer I, white matter, or cerebellar cortex.
What’s more, the researchers discovered that the sequence of anti-sense DPP10 RNA has GC-rich areas that could allow it to form a stem-loop structure to interact with Polycomb 2 and transcription start sites — qualities that would support the RNA’s role in regulation.
Now, there’s no disease known to result from any DPP10 epigenetic modifications. But the authors note that rare variants “confer strong genetic susceptibility to autism, while some of the gene’s more common variants contribute to a significant risk for bipolar disorder, schizophrenia, and asthma.” That is, it’s a pretty good candidate for a gene that affects cortex function: Also according to the authors, it encodes a dipeptidyl peptidase-related protein that regulates potassium channels and neuronal excitability.
(It goes beyond DPP10, too. Five other genes associated with some of the 33 human-specific H3K4me3 peaks have ties to psychiatric diseases.)
So does all this point to an epigenetic role in human brain evolution? It starts to look even more convincing, considering what the researchers report about genetic changes in the DNA around these H3K4me3 marks, since the DNA sequence might very well have an important influence on histone binding and other functions. Compared to macaques, chimps, gorillas, and orangutans, the human versions of these DNA sequences have undergone major changes, with nucleotide substitution rates of 2- to 5-fold.
Meanwhile, nearby protein-coding sequences remained relatively unchanged in humans, as compared with the other primates. Also, compared to our close hominid relatives Homo denisova and Neanderthals, the nucleotide substitution rate near human H3K4me3 is much lower — about half the rate that the comparisons with non-human primates revealed. So the authors speculate that …
Taken together, these results suggest that at least a subset of the TSS regions with H3K4me3 enrichment in human (compared to non-human primates) were exposed to evolutionary driven DNA sequence changes on a lineage of the common ancestor of H. sapiens and the archaic hominins, but subsequently were stabilized in more recent human evolution, after splitting from other hominins.
What I like about this research is that the team used real human brain cells. No, not from living people, of course. Cadavers. And they didn’t just grind up a lump of brain in the blender, either. They painstakingly separated prefrontal cortex neurons from glial cells and other types to get at the real differences in the cells that matter, from the brain region that makes executive decisions and fancy associations.
What’s more, they may have sidestepped the problem with dead tissue — sample degradation – by measuring histone methylation. Apparently these marks don’t appear to change much after their host has died.
What I don’t like about the study is pretty standard. The human brain samples came from only 11 subjects — seven children and four adults.
The research team focused only on H3K4me3 peaks that all the humans had in common, so although they might’ve missed some peaks, it’s not that bad of a shortcoming. But there’s also the problem that the human subjects’ brains had already developed — as the authors hint in their discussion, fetal neuronal gene regulation could hold many of the important secrets, since the period of actual brain development is when you’ll probably find a lot of human-specific differences.
I’m anxious to see more of these kinds of studies, and I bet they’re in the works right this minute. It seems that there’s finally a big enough set of lab tools to get at the nitty-gritty parts of big questions.
[Flickr user IsaacMao's picture "Child Brain" is used here under a Creative Commons license.]
Shulha HP, Crisci JL, Reshetov D, Tushir JS, Cheung I, Bharadwaj R, Chou HJ, Houston IB, Peter CJ, Mitchell AC, Yao WD, Myers RH, Chen JF, Preuss TM, Rogaev EI, Jensen JD, Weng Z, & Akbarian S (2012). Human-specific histone methylation signatures at transcription start sites in prefrontal neurons. PLoS biology, 10 (11) PMID: 23185133
When it comes to acetylation and epigenetics your mind probably goes right to histones. Acetylated histones are associated with relaxed, transcriptionally active DNA. However, acetylation is an important post-translational modification of lysine in many cellular proteins. It is as widespread as phosphorylation. It is reversable. Functionally, acetylation is known to be involved in the effects of calorie restriction on metabolism and aging. Now the first direct evidence of a mechanism underlying this process has been reported.
The journal Molecular Cell has recently published Calorie Restriction and SIRT3 Trigger Global Reprogramming of the Mitochondrial Protein Acetylome, authored by scientists from the University of Wisconsin-Madison and the University of Tokyo. They used model mice with age-related hearing loss for this study. This hearing loss is prevented by calorie restriction (CR). They explored the liver mitochondrial protein acetylome by developing a new quantitative, acetyl-proteomic method. The Sirtuin family of deacetylases were already linked to CR. And Sirtuin3 was recently shown to be induced during CR. So the researchers explored the regulatory role of tSIRT3 during CR. They identified multiple pathways where SIRT3 is involved in reprogramming mitochondria during CR via acetylation. Interestingly for “Epiexperts”, among those ID’d were mitochondria DNA expression and maintenance pathways. Part of that SIRT3 led CR reprogramming, is through regulation of mtDNA transcription factors, mtRNA polymerases and mitochondrial ribosome proteins. Please see Table 1. in the pub for the list of SIRT3 targets in this functional group. The reprogramming during CR of these targets leads to enhanced mtDNA transciption, mtDNA translation and protein quality control.
Recall, mitochondria function to produce ATP from sugar and oxygen, powering the cell. This process also creates oxidative free radicals that contribute to aging. Mitochondria have their own DNA and can make their own proteins. During calorie restriction, mitochondria performance is actually enhanced. They use less oxygen and produce less damaging free radicals. Again, in the model mice this results in prevention of hearing loss.
This is a great paper worth reading. Mainly because it establishes that SIRT3, (of the sirtuins deacetylases family) is directly linked to anti-aging effects. But also because it shows SIRT3 is potentially a significant regulator of mitochondrial epigenetics. It’s like by eating less, SIRT3 directs mitochondria to become environmentally friendly power stations with high energy efficiency ratings.
Hebert AS, Dittenhafer-Reed KE, Yu W, Bailey DJ, Selen ES, Boersma MD, Carson JJ, Tonelli M, Balloon AJ, Higbee AJ, Westphall MS, Pagliarini DJ, Prolla TA, Assadi-Porter F, Roy S, Denu JM, & Coon JJ (2012). Calorie Restriction and SIRT3 Trigger Global Reprogramming of the Mitochondrial Protein Acetylome. Molecular cell PMID: 23201123
Microbiologists rushed to respond to the 2011 pathogenic E.coli (0104:H4) outbreak in Europe. The new strain’s DNA was sequenced within 3 days time. The trace back investigation identified an organic bean sprouts field as the source. Now, Pacific Biosciences with collaboration from New England Biolabs, reports Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing in the journal, Nature Biotechnology (open access paper).
Epigenetic analysis reveals the potential for restriction modification methyltransferase enzymes (RM MTases) to have important roles in this pathogenic phenotype. 0104:H4 phenotype virulence has been defined by its production of high levels of Shiga toxin. AND it turns out that this strain has specific MTases that can promote that production.
SMRT sequencing was used to simultaneously map both the E.coli DNA sequence and strand-specific methylation patterns, yielding functional information. From this data sequence motifs for strain specific methyltransferase enzymes were deduced. New MTase activities were ID’d by correlating the methylation patterns with transcriptome profiles. The functions of these MTases were probed. In one experiment when a phage encoding both the shiga toxin and an 0104:H4 MTase was incorporated into the genome of a non-pathogenic E.coli strain, 1/3 of its transcriptome changed and it was able to produce the Shiga toxin. In another experiment, knocking out a 0104:H4 specific MTase from the strain showed significant epigenetic changes affecting multiple systems.
It’s impressive how the third generation SMRT sequencing technology can be applied to discovering bacterial epigenetic processes key to establishing pathogenicity. Hopefully these techniques will enable the development of new medicines for hemorrhagic colitis and hemolytic uremic syndromes to save lives!
Fang G, Munera D, Friedman DI, Mandlik A, Chao MC, Banerjee O, Feng Z, Losic B, Mahajan MC, Jabado OJ, Deikus G, Clark TA, Luong K, Murray IA, Davis BM, Keren-Paz A, Chess A, Roberts RJ, Korlach J, Turner SW, Kumar V, Waldor MK, & Schadt EE (2012). Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing. Nature biotechnology PMID: 23138224
The most recent pub from the stream of research put forth by New England Biolabs scientists, is a collaboration with scientists from Pacific Biosciences™ . See this open access paper Iain A. Murray et al. The methylomes of six bacteria. (2012) Nucleic Acids Research. It demonstrates how the 3rd generation SMRT DNA sequencing system is used to explore bacterial methylomes. Many exciting discoveries about microbe epigenetic systems are sure to follow this technological advance!
So why is DNA methylated in bacteria? Mainly it functions as part of restriction modification systems. But bacterial methyltransferases also take part in gene expression, host-pathogen interactions, DNA damage, and DNA repair. Microbe methylation modifications include N6-methyladenine (6-mA), N4-methylcytosine (4-mC) & 5-methylcytosine (5-mC).
Single-molecule, real-time sequencing, or SMRT, analyzes DNA as it is sequenced by collecting light pulses emitted as a byproduct of labelled nucleotide incorporation. Algorithmic analysis is subtle enough to detect certain base modifications. A caveat to methylation analysis is that 5-mC is still a bit too subtle. However, for this study, associated methyltransferase genes were cloned and analyzed separately, where appropriate, to ID 5mC sites. The researchers were able to perform methyltransferase (MTase) recognition motif analysis. The recent upgrade of the PacBio® RS High Resolution Genetic Analyzer software also made this project possible.
Richard Roberts, Ph.D., New England Biolab’s Chief Scientific Officer, and senior author on the paper, commented: “DNA methylation is widespread in bacteria where it can protect against restriction enzymes and also regulate gene expression. Until the advent of SMRT sequencing it was not possible to examine the complete methylome of any bacterium. Now it has become simple and we are awash in fascinating new data. Understanding the biological significance of these methylation patterns represents a welcome new challenge for microbiologists.”
I’m sure all you Epiexperts are just as keen to see how the data on microbe gene expression, controlling functions like adaptability and disease pathology, plays out.
Murray IA, Clark TA, Morgan RD, Boitano M, Anton BP, Luong K, Fomenkov A, Turner SW, Korlach J, & Roberts RJ (2012). The methylomes of six bacteria. Nucleic acids research PMID: 23034806