Epigenetic Natural Variation in Arabidopsis thaliana


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A new research paper appearing today in PLoS Biology provides new insight into the DNA methylation patterns in the plant Araabidopsis thaliana:

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F2 families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.

Link

CpG Methylation Targeted to Transcription Units in an Invertebrate Genome


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I just came across an interesting research paper by Suzuki et al. in a recent issue of Genome Research: CpG methylation is targeted to transcription units in an invertebrate genome.

DNA is methylated at the dinucleotide CpG in genomes of a wide range of plants and animals. Among animals, variable patterns of genomic CpG methylation have been described, ranging from undetectable levels (e.g., in Caenorhabditis elegans) to high levels of global methylation in the vertebrates. The most frequent pattern in invertebrate animals, however, is mosaic methylation, comprising domains of methylated DNA interspersed with unmethylated domains. To understand the origin of mosaic DNA methylation patterns, we examined the distribution of DNA methylation in the Ciona intestinalis genome. Bisulfite sequencing and computational analysis revealed methylated domains with sharp boundaries that strongly colocalize with 60% of transcription units. By contrast, promoters, intergenic DNA, and transposons are not preferentially targeted by DNA methylation. Methylated transcription units include evolutionarily conserved genes, whereas the most highly expressed genes preferentially belong to the unmethylated fraction. The results lend support to the hypothesis that CpG methylation functions to suppress spurious transcriptional initiation within infrequently transcribed genes.

The article is freely accessible to all. Link

DNA Methylation Affects Nuclear Organization and Histone Modifications


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In a paper recently published in the Journal of Cell Biology, Gilbert et. al. use mutant mouse embryonic stem cells lacking DNA methylation to show that DNA methylation affects nuclear organization and nucleosome structure, but not chromatin compaction.

DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.

References:

Gilbert N, Thomson I, Boyle S, Allan J, Ramsahoye B, Bickmore WA. 2007. DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction. Journal of Cell Biology 177(3):401-411.
doi:10.1083/jcb.200607133

Moving Towards Better Mapping of CpG Islands


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A new research paper available as an early online release in the journal PLoS Compitational Biology helps provide some new insight into how the definition and mapping of CpG islands could be improved.

CpG islands were originally identified by epigenetic and functional properties, namely absence of DNA methylation and frequent promoter association. However, this concept was quickly replaced by simple DNA sequence criteria, which allowed for genome-wide annotation of CpG islands in the absence of large-scale epigenetic datasets. Although widely used, the current CpG island criteria incur significant disadvantages: (i) reliance on arbitrary threshold parameters which bear little biological justification; (ii) failure to account for widespread heterogeneity among CpG islands; and (iii) apparent lack of specificity when applied to the human genome. This study is driven by the idea that a quantitative score of “CpG island strength”, which incorporates epigenetic and functional aspects, can help resolve these issues. We construct an epigenome prediction pipeline that links the DNA sequence of CpG islands to their epigenetic states, including DNA methylation, histone modifications, and chromatin accessibility. By training support vector machines on epigenetic data for CpG islands on human chromosomes 21 and 22 we identify informative DNA attributes that correlate with open and compact chromatin structures, respectively. These DNA attributes are used to predict the epigenetic states of all CpG islands genome-wide. Combining predictions for multiple epigenetic features we estimate the inherent CpG island strength for each CpG island in the human genome, i.e. its inherent tendency to exhibit an open and transcriptionally competent chromatin structure. We extensively validate our results on independent datasets, showing that the CpG island strength predictions are applicable and informative across different tissues and cell types, and we derive improved maps of predicted “bona fide CpG islands”. The mapping of CpG islands by epigenome prediction is conceptually superior to widely used CpG island criteria since it links CpG island detection to their characteristic epigenetic and functional states. And it is superior to purely experimental epigenome mapping for CpG island detection since it abstracts from specific properties that are limited to a single cell type or tissue. In addition, using computational epigenetics methods we could identify strong correlation between the epigenome and characteristics of the DNA sequence, which emphasizes the need for a better understanding of the mechanistic links between genome and epigenome.

Link

Illumina Launches Custom Methylation Application


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Today Illumina, Inc. (NASDAQ:ILMN) launched a custom-content methylation product that allows researchers to perform methylation profiling specific to individual CpG sites. From the press release:

Joining Illumina’s GoldenGate Methylation Cancer Panel I, the Company’s first standard methylation product launched in January 2007, investigators now have the option to select their favorite genes or gene regions to cost-effectively survey up to 1,536 methylation sites of choice across 96 samples simultaneously.

“Custom methylation adds another degree of integration to analysis with genetic and epigenetic data. Researchers who are interested in expediting the speed and scope of their methylation studies in areas such as cancer, developmental disorders, stem cell, aging, and neurological diseases now have the ability to select specific CpG sites from chosen genes” said Marina Bibikova, Ph.D., Staff Scientist at Illumina.

Link

Epigenetics in Focus at Nature Reviews Genetics


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Following closely on the heels of the special issue of Cell on epigenetics, Nature Reviews Genetics today published its own focus issue on epigenetics (April 2007), with reviews from some of the most prominent experts in several sub-disciplines within epigenetics, including stem cell research, cancer epigenomics, and environmental epigenetics. The editors of the journal open the issue with a brief introduction:

The explosion of interest in epigenetics over the past few years has had an impact on many branches of genetic and genomic research. One of the hottest topics in the field of gene regulation relates to the role of epigenetic modifications in dictating the expression output of the genome. In genomics, the advent of technologies for the large-scale profiling of these marks has made the characterization of epigenomes a coveted goal. And chromosome biologists are increasingly learning how epigenetic modifications contribute to the structural packaging of the genetic material at various levels.

The cover of the issue, pictured below, is a cartoon sketchboard by the journal’s art editor, Patrick Morgan.

The issue is packed with five reviews on epigenetics topics of interest:

  • Environmental epigenomics and disease susceptibility by Jirtle RL and Skinner MK. Epigenetic modifications provide a possible link between the environment and disease-causing alterations in gene expression. Evidence from animal studies increasingly supports this theory, including recent findings of epigenetically mediated transgenerational alterations in phenotype that are caused by environmental exposure. Link
  • Epigenetic signatures of stem-cell identity by Spivakov M and Fisher AG. How do stem cells keep the genes that drive differentiation in a repressed state, while maintaining the ability to express them in the future? Increasing evidence indicates that distinctive epigenetic traits underlie this unique aspect of stem-cell biology. Link
  • Transposable elements and the epigenetic regulation of the genome by Slotkin RK and Martienssen R. Cells use a range of increasingly well understood epigenetic mechanisms to keep transposable elements under control. These silencing mechanisms have been co-opted during the course of evolution to contribute to key aspects of chromosome biology and gene regulation. Link
  • Cancer epigenomics: DNA methylomes and histone-modification maps by Esteller M. Recent technological advances allow epigenetic alterations in cancer to be studied across the whole genome. These approaches are being used to answer key outstanding questions about cancer biology, and to provide new avenues for diagnostics, prognostics and therapy. Link
  • The epigenetic regulation of mammalian telomeres by Blasco MA. Epigenetic modifications are key players in the regulation of fly and yeast telomeres, and recent studies indicate that the same applies in mammalian cells. These findings have implications for our understanding of the roles of telomeres in ageing and cancer. Link

The recent surge of coverage by high impact journals in the area of epigenetics likely reflects the major advances and discoveries made in recent years, and will hopefully provide renewed interest among scientists, funding bodies, and most importantly, the general public.

DNA Methylation Involved in Memory Formation


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Researchers at the University of Alabama at Birmingham have published new research supporting their hypothesis that DNA methylation plays a role in forming memories (1). The link between methylation and memory formation came about from the observation that methylation was disregulated in people suffering from brain disorders such as autism and schizophrenia.

In their experiments, the researchers created fearful memories in rats by placing them in specific training chambers that gave them a mild shock. They could then test whether the rat remembered the shock by observing if the rat froze when placed in the same chamber.

The researchers used methylation inhibitors to discover that DNA methylation directly controlled the activity of genes known to either suppress or promote memory formation.

“To our knowledge, this study is the first to present evidence that DNA methylation, once thought to be a static process after cellular differentiation, is not only dynamically regulated in the adult nervous system but also plays an integral role in memory formation,” concluded Miller and Sweatt. They wrote that their findings indicate that DNA methylation has been co-opted by the central nervous system as a “crucial step” in regulating gene activity involved in memory formation.

The study is available in the March 15, 2007 edition of the journal Neuron. Link

References:

Miller CA and Sweatt JD. 2007. Covalent modification of DNA regulates memory formation. Neuron 53:857-869.
DOI 10.1016/j.neuron.2007.02.022.

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Liposuctioned Fat as a Source of Stem Cells


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In 2004, members of the American Society of Plastic Surgerons performed over 320,000 liposuction procedures (1). Who knew that the extracted fat was a potential source of stem cells for research or therapeutics.

Dr. Philipe Collas at the University os Oslo in Norway is conducting research to identify the stem cells among liposuctioned fat cells that are the best at regenerating tissue.

“Fat tissue is an underappreciated source of stem cells,” Collas pointed out. Unlike other sources of adult stem cells, such as bone marrow, fat is abundant and there is no shortage of donors. “It’s wonderful, we have litres and litres of material from cosmetic surgery clinics and end up with bucketfuls of stem cells to work with,” he notes.

Researchers ackowledge that the key to transforming adult stem cells from fat into other cell types is in their epigenetic signature, such as the level of methylation.

Epigenetic marks contribute to switching genes on and off, and stem cells rely on them heavily as they divide and mature. The Oslo team has found that low rates of DNA methylation, for instance, boost the chances of transforming fat stem cells from one cell type into another. “Look at a cell’s epigenetic profile,” says Collas, “and you may be able to predict what that cell is likely to turn into.”

These epigenetic signatures have grabbed everyone’s attention, acknowledges Ernest Arenas, a EuroSTELLS researcher at the Karolinska Institute in Stockholm, Sweden. “Scientists in the stem cell field are starting to realise that for cell manipulations to succeed they need to pay attention to their epigenetic marks. Cells can’t be pushed along to become a different cell type unless they start out with the right set of [epigenetic] conditions.”

Epigenetics remains one of the most promising avenues of research for identifying ways to differentiate plentiful adult stem cells into other cell types for therapeutic purposes. Link

Gadd45a Promotes Epigenetic Gene Activation by Repair-Mediated DNA Demethylation


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Nature has published a letter from researchers at the German Cancer Research Center involving their implication of the gene Gadd45a in one of the black boxes of epigenetic mechanisms: demethylation.

DNA methylation is an epigenetic modification that is essential for gene silencing and genome stability in many organisms. Although methyltransferases that promote DNA methylation are well characterized, the molecular mechanism underlying active DNA demethylation is poorly understood and controversial. Here we show that Gadd45a (growth arrest and DNA-damage-inducible protein 45 alpha), a nuclear protein involved in maintenance of genomic stability, DNA repair and suppression of cell growth, has a key role in active DNA demethylation. Gadd45a overexpression activates methylation-silenced reporter plasmids and promotes global DNA demethylation. Gadd45a knockdown silences gene expression and leads to DNA hypermethylation. During active demethylation of oct4 in Xenopus laevis oocytes, Gadd45a is specifically recruited to the site of demethylation. Active demethylation occurs by DNA repair and Gadd45a interacts with and requires the DNA repair endonuclease XPG. We conclude that Gadd45a relieves epigenetic gene silencing by promoting DNA repair, which erases methylation marks.

Link

One of the experiments from this paper seem to support the recent finding that demethylation of the proximal-promoter region is required for active transcription.

ScienceDaily offers a summary of this research adapted from a press release from the German Cancer Research Center. Link

Cell Reviews Epigenetics and Chromatin Organization


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The journal Cell has released a special review issue, “Epigenetics and Chromatin Organization.” The issue contains 11 review articles, beginning with a review of one of the most exciting aspects of epigenetics: its effect on evolution.

According to classical evolutionary theory, phenotypic variation originates from random mutations that are independent of selective pressure. However, recent findings suggest that organisms have evolved mechanisms to influence the timing or genomic location of heritable variability. Hypervariable contingency loci and epigenetic switches increase the variability of specific phenotypes; error-prone DNA replicases produce bursts of variability in times of stress. Interestingly, these mechanisms seem to tune the variability of a given phenotype to match the variability of the acting selective pressure. Although these observations do not undermine Darwin’s theory, they suggest that selection and variability are less independent than once thought. Link

A nice summary of this review article is available at Gene Expression.

Cell

The other review articles include:

This special review issue from Cell is a clear indication of the role that epigenetics is playing in changing the scope and direction of scientific research in many different areas. My hope is that epigenetics will continue to inspire more articles in the press and will become well known among both those in science and the general public. Link

Unmethylated Promoter-Proximal Region Required for Transcription


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A new paper published this week in PLoS Genetics provides evidence that an unmethylated region extending several hundred base pairs from the promoter of a gene is required for activation of transcription.

Genes, the functional units of heredity, are made up of DNA, which is packaged inside the nuclei of eukaryotic cells in association with a number of proteins in a structure called chromatin. In order for transcription, the process of transferring genetic information from DNA to RNA, to take place, chromatin must be decondensed to allow the transcription machinery to bind the genes that are to be transcribed. In mammals, promoters, the starting position of genes, are frequently embedded in “CpG islands,” regions with a relatively high density of the CpG dinucleotide. Paradoxically, while cytosines in the context of the CpG dinucleotide are generally methylated, CpGs flanking the start sites of genes typically remain methylation-free. As CpG methylation is associated with condensed chromatin, it is generally believed that promoter regions must remain free of methylation to allow for binding of the transcription machinery. Here, using a novel method for introducing methylated DNA into a defined genomic site, we demonstrate that DNA methylation in the promoter-proximal region of a gene is sufficient to block transcription via the generation of a chromatin structure that inhibits binding of the transcription machinery. Thus, methylation may inhibit transcription even when present outside the promoter region.

PLoS Genetics is an open access journal with free access to full text articles. Link

Optimizing Annealing Temperature in Bisulfite Methylation Analysis


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Sodium bisulfite treatment of DNA is widely used by researchers to analyze the DNA methylation patterns of DNA regions of interest. With bisulfite treatment of DNA, researchers are able to quantitatively determine if a 5′ cytosine is methylated. If a cytosine (C) is methylated, bisulfite treatment will leave the cytosine untouched. If the cytosine is unmethylated, bisulfite treatment will convert the C to a uracil (U). Researchers can then sequence a region of interest after bisulfite treatment to determine the 5′ cytosines that are methylated and unmethylated.

A new paper in the January 2007 issue of BioTechniques shows that PCR bias can be reduced by optimizing the annealing temperature in PCR methylation analysis (1).

The main concern for PCR-based quantitative DNA methylation analysis is PCR bias, which is due to the fact that methylated and unmethylated DNA molecules sometimes amplify with greatly differing efficiencies.

After analysis of several variables in the PCR reaction mixture that could contribute to this variability, the authors concluded that increasing annealing temperature in the PCR reaction resulted in a higher level of methylation detected.

It is clear from the results that regardless of the primer system, there is a strong bias toward the amplification of unmethylated DNA, and increasing annealing temperature for PCR improved the amplification toward methylated DNA.

References:

1. Shen L, Guo Y, Ahmed S, Issa JJ. 2007. Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. BioTechniques 42(1):48-58.

Histone Modifications Regulate Pluripotency in the Early Mouse Embryo


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Earlier this year, a group affiliated with Cambridge University at the Wellcome Trust/Cancer Research UK Gordon Institute reported in Nature that epigenetics, specifically methylation of certain arginine residues of histone H3, directly contribute to cell fate and success in the four-cell stage embryo in the mouse model. These findings confront the widely accepted paradigm that mammalian embryos begin development with similar, if not identical, cell types which differ only when inside and outside cells form. Furthermore, the findings solidify that epigenetic modifications influence cell direction and determination.

By investigating the belief that epigenetic mechanisms are utilized to support pluripotency, the researchers provided evidence that arginine methylation of histone H3 is at its highest in four-cell blastomeres which contribute to the inner cell mass (ICM), polar trophectoderm and fully develop when joined with chimaeras. Inversely, arginine methylation of histone H3 is lowest in cell progeny which contribute primarily to mural trophectoderm which exhibit abnormal development when joined with chimaeras. This finding indicates that maximal levels of arginine methylation of histone H3 influence blastomeres’ contribution to pluripotent cells of the inner cell mass. Furthermore, over-expression of the histone H3 arginine methyltransferase gene CARM1 in blastomeres resulted in direction of subsequent progeny cells to the ICM – solidifying the theory that “specific histone modifications are the earliest known epigenetic marker contributing to development of ICM” and precede formation of inside and outside cells.

References:

Torres-Padilla ME, Parfitt DE, Kouzarides T, Zernicka-Goetz M. 2007. Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature 445:214-218.
doi:10.1038/nature05458

Transgenerational Epigenetic Modification with Nutritional Supplementation


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Reader Israel Barrantes recently commented on what he considered to be the “most memorable epigenetic moment of the year” for 2006, which was a groundbreaking paper by Cropley et al. that appeared in Proceedings of the National Academy of Science in November (1). I couldn’t argue with that nomination, so I decided to write about the paper to kick off the week of Just Science.

The paper, titled “Germ-line epigenetic modification of the murine Avy allele by nutritional supplementation,” uses a mutant mouse strain known as viable yellow agouti, or Avy. As shown previously, mice carrying the viable yellow agouti allele exhibit yellow fur, obesity, type II diabetes, and predisposition to tumors. Those that carry one Avy allele and one normal allele (referred to as Avy/a) exhibit varying degrees of the Avy phenotype, ranging from fully yellow and obese to lean and fully agouti. In previous studies, it has been shown that pregnant Avy females that receive a diet supplementation containing folate, choline, betaine, and vitamin B12 from two weeks prior to gestation to birth produce Avy offspring that are shifted toward the agouti phenotype. This shift was also highly correlated with an increase in cytosine methylation. In other words, nutritional supplementation during gestation seemed to cause an epigenetic alteration in phenotypes of offspring.

The picture below shows samples of the varying degrees of the yellow to agouti mice and their corresponding scores. The scores are used in this study as a quantitative way of determining the degree of tranmission of the Avy allele, comparing results when the mutant allele is contributed by the male (sire) or female (dam). The authors found that the previously mentioned shift to the agouti phenotype occurred only when the Avy allele was contributed by the sire, which provides evidence that the male germ line may play a role in transgenerational epigenetic alterations.

Agouti Mice

The authors then used this evidence of male-specific transmission of the Avy allele to propose that the altered phenotype could be passed to a subsequent generation without further diet supplementation. Further, they wanted to determine if diet supplementation was required throughout gestation to induce the epigenetic alteration. The authors proposed that supplementation was only critical during the period encompassing the point at which primordial germ cells differentiate and reset epigenetic marks. Therefore, the period of supplementation for pregnant a/a dams mated to Avy/a sires was set at E8.5 (embryonic day 8.5, or 8.5 days past conception) to E15.5. (Gestation in mice is about 21 days.) Interestingly, this midgestation exposure was very similar to the timepoint used in another study identifying a transgenerational epigenetic effect in mammals (2).

The authors found that when the F1 generation whose mothers received diet supplementation during gestation were mated, the F2 generation exhibited a similar shift in color score as the F1 generation. It is worth emphasizing that the F2 generation embryos were not directly exposed in utero to diet supplementation as the F1 generation embryos were, but the germ line of F2 animals was affected by the diet supplementation given to the previous generation.

This study was groundbreaking in that it provides the first direct evidence of a mechanism in a transgenerational, epigenetic alteration. However, it would be interesting to see if the shift to the agouti phenotype would continue into the F3 and F4 generations, as would be expected if the epigenetic germ line was permanently reprogrammed.

References:

1. Cropley JE, Suter CM, Beckman KB, Martin DIK.
2006. Germ-line epigenetic modification of the murine Avy allele by nutritional supplementation. Proc Natl Acad Sci USA 103:17308-17312.
doi:10.1073/pnas.0607090103
2. Anway MD, Cupp AS, Uzumcu M, Skinner MK. 2005. Epigenetic Transgenerational Actions of Endocrine Disruptors and Male Fertility. Science 308:1466-1469.
doi:10.1126/science.1108190

Epigenetic Control of Regulatory T Cells


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New research published this week in PLoS Biology shows the importance of epigenetic modifications in regulatory T cells.

Regulatory T cells play a pivotal role in the maintenance of self-tolerance within the immune system by preventing autoimmunity or excessive activation of the T cells that respond to pathogens (naïve and effector T cells). They differentiate within the thymus, but can also be de novo induced in the rest of the body. Mechanisms determining development of a stable regulatory T cell lineage are unknown. Our study provides evidence for a critical role of epigenetic modifications in the locus coding for the forkhead transcription factor Foxp3, which acts as a master switch controlling regulatory T cell development and function: An evolutionarily conserved region within the non-coding part of the gene contains CpG motifs, which are completely demethylated in regulatory T cells, but methylated in naïve and effector T cells, whereas we observed an inverse occurrence of acetylated histones, another epigenetic chromatin modification. Regulatory T cells induced in vitro—which, in contrast to natural regulatory T cells, do not display a stable regulatory T cell phenotype—display only incomplete DNA demethylation despite high Foxp3 expression. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to result in a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of regulatory T cells and for the understanding of long-term cell lineage decisions.

Link