EpiGenie | Epigenetics, Stem Cell, and Synthetic Biology News http://epigenie.com Scientific News, Technology, and Product Information Fri, 16 Mar 2018 18:49:06 +0000 en-US hourly 1 https://wordpress.org/?v=4.9.4 Cutting Through the Epigenetic Complexities Linking Aging and Tumorigenesis http://epigenie.com/cutting-epigenetic-complexities-linking-aging-tumorigenesis/ http://epigenie.com/cutting-epigenetic-complexities-linking-aging-tumorigenesis/#respond Fri, 16 Mar 2018 14:40:07 +0000 http://epigenie.com/?p=26927 Writing your first grant proposal? Optimizing that tricky Western blot? Programming an old PCR machine? Only a few things are more complex than our current understanding of the epigenetic links between aging and tumorigenesis, but this has not stopped a few valiant labs from doubling down and busting a few paradigms while they are at it!

In general, aging is the most important risk factor for cancer development; the older you are, the more likely you are to suffer from this often-devastating disease. But why? One hypothesis states that the accumulation of epigenetic alterations over time predisposes our cells to go rogue, but quite how they do it is open to interpretation. In a related hypothesis, the epigenetic profile of the senescent cells that accumulate in the aging body may foster tumorigenicity if cells can escape the senescent state. Furthermore, we also know that focal DNA hypermethylation and global DNA hypomethylation occur in both tumorigenesis and aging/senescence, suggesting an epigenetic link between the two processes.

Now, two studies from laboratories from opposite sides of the Atlantic have cut through some of the epigenetic complexity surrounding the link between aging and tumorigenesis to provide fresh insight into this highly complicated relationship.

Same Old DNA Hypomethylation, Different Chromatin Context!

Our first study from the labs of Agustín F. Fernández and Mario F. Fraga (Universidad de Oviedo, Spain) aimed to fill a few knowledge gaps concerning DNA hypomethylation during aging and tumorigenesis. Previous studies have discerned a robust link between the chromatin patterns of DNA hypermethylation sites in tumorigenesis and aging, although few have reported on the chromatin context of DNA hypomethylation. This prompted the authors to integrate 450K array data generated by The Cancer Genome Atlas consortium from 2,311 healthy and tumoral samples obtained from differentially aged individuals with histone, chromatin state, and transcription factor binding site data from the NIH Roadmap Epigenomics and ENCODE projects.

After the computational processing winded down and the dust settled, Pérez and colleagues discovered:

  • Hyper- and hypo-methylated changes display a similar distribution throughout the genome but also exhibit some tissue-independent alterations
    • Bidirectional changes in DNA methylation occur during tumorigenesis, while hypermethylation predominates during aging
    • Interestingly, the authors identified common DNA methylation signature that occurred across all tumors, and, separately, a common DNA methylation signature in many aged tissues
    • Most tumor types do not exhibit age-associated DNA methylation changes, which is in agreement with the reprogramming of the epigenetic clock in cancer cells
  • As expected, hypermethylated regions associate with both aging and tumorigenesis display chromatin modifications characteristic of bivalent chromatin domains
    • Hypermethylation changes tend to occur in CpG-dense regions, associated with binding of the EZH2 and SUZ12 polycomb components, and affect developmentally-associated genes
  • However, hypomethylated DNA sequences associate with different chromatin contexts during aging and tumorigenesis
    • During aging, DNA hypomethylated sequences occur at enhancers with the activating H3K4me1 modification
    • During tumorigenesis, DNA hypomethylation arises at heterochromatic sites displaying the repressive H3K9me3 modification
    • While hypomethylated regions in tumorigenesis affect genes associated with cellular signaling, the study observed no strong correlations for aging

What does this all mean? The data gathered here suggests that while the story may appear straightforward for DNA hypermethylation, DNA hypomethylation occurs in different chromatin contexts during tumorigenesis and aging, suggesting that different mechanisms may be at play.

DNA Methylation Profiling: Busting Cancer Paradigms and Tracking Cancer Risks

Our second study from the labs of Stephen B. Baylin and Hariharan Easwaran (Johns Hopkins University, Baltimore, USA) employed DNA methylation profiling to investigate the hypotheses that tumor-promoting epigenetic states arise in the senescent cells that accumulate in the aging body. Xie and colleagues compared DNA methylation alterations during the tumorigenic transformation of engineered fibroblasts and replicative senescence of unmodified fibroblasts with the 450K array.

Their new work not only busts a cancer paradigm, but may also provide a new means to stratify cancer patients into risk categories:

  • While DNA methylation patterns during tumorigenesis and senescence appear globally similar, transformation induces a significantly different DNA methylation state when compared to senescence
  • Senescence-associated promoter hypermethylation events mainly involve the silencing of biosynthesis- and metabolism-associated genes in cells unlikely to undergo transformation
    • The shutdown of biosynthetic processes via DNA methylation likely inhibits tumor development, thereby representing an epigenetic contribution to the Hayflick phenomenon
  • Methylation changes during transformation arise stochastically at pro-survival and developmental gene promoters and occur independently of senescence-associated changes
    • Many of these genes display promoter hypermethylation in primary tumors
  • Of note, the study discovered a subset of self-renewal and cell survival genes commonly methylated during tumorigenic transformation and senescence
    • These genes represent hotspots for DNA hypermethylation in primary tumors and aging tissues and, perhaps, a crucial means to track cancer risk

Overall, these new findings do not support a role for senescence-associated methylation in tumorigenesis but do support a role for the random DNA hypermethylation events that accumulate during normal aging; specifically, patients who accrue DNA methylation at select gene promoters may present with an increased likelihood of tumorigenesis.

Conclusions: Complexity Cut?

As with many good studies, cutting through the epigenetic complexities has redefined some of our basic assumptions regarding the links between aging and cancer and provided further engaging questions moving forward. Future research may uncover the different mechanisms controlling DNA hypomethylation in during aging/tumorigenesis and refine the use of DNA methylation analysis as a potent means to define cancer risk.

For more on the complexities of DNA hypomethylation events during tumorigenesis and aging, see Aging Cell, March 2018, and for the low down on DNA methylation and cancer risk, head on over to Cancer Cell, February 2018.

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Epigenetics Meets Cartography: Precision Mapping of Single Nucleosomes and Linkers http://epigenie.com/epigenetics-meets-cartography-precision-mapping-single-nucleosomes-linkers/ http://epigenie.com/epigenetics-meets-cartography-precision-mapping-single-nucleosomes-linkers/#respond Thu, 15 Mar 2018 02:03:52 +0000 http://epigenie.com/?p=26922 While we’ve asked before how nucleosomes find their way to the right spot on DNA, sometimes we’re left staring at sea monsters in the most tantalizing unexplored areas on our genome-wide maps. When it comes to nucleosomes, positioning is everything since it affects DNA accessibility for replication, repair, and gene expression. In order to understand how nucleosome positions are determined, we need a reliable way to map them. Previously, nucleosome mapping has relied upon H4S47C cleavage, which utilizes chemical cleavage at a mutated cysteine (S47C) in histone 4 (H4), but unfortunately it produces high background noise. Luckily, a talented team from the lab of of Steven Henikoff at the Fred Hutchinson Cancer Research Center have developed a new technique for precision mapping of nucleosomes in the yeast genome.

The authors have improved upon the H4S47C cleavage mapping method by relocating the cysteine mutant to residue 85 (H85C) on histone 3 (H3). Both methods exploit the ability of phenanthroline to chelate copper to the cysteine residue. Addition of peroxide produces free radicals at the conjugated copper, resulting in adjacent DNA backbone cleavage. The resulting DNA fragments are then sequenced. Although H3Q85C and H4S47C cleavage mapping operate on the same principles, H3Q85C has several advantages over its parental method:

  • The H3Q85C cleavage site is further from the center of the nucleosome
    • A 51-base pair fragment is released upon cleavage, which is long enough for sequencing
  • The center of each 51-base pair fragment directly corresponds to nucleosome position and consequently, H3Q85C mapping does not rely on predictive averages
  • There is less cleavage within linker regions and reduced non-specific cleavage, resulting in a higher signal-to-noise ratio

These ambitious authors weren’t content to simply develop this improved technique for nucleosome mapping—they immediately put this new methodology to work. Here’s a look at how they applied this powerful technique:

  • H3Q85C mapping can detect H3 containing nucleosomes
    • This feature was used to disprove a previous hypothesis of a Cse4-H3 heterotypic nucleosome at yeast centromeres
    • They also showed that tRNA genes are depleted of nucleosomes
  • The improved signal-to-noise ratio allowed for identification of rotational phasing across all nucleosomes, confirming the preference for A/T dinucleotides in the minor groves of the DNA that contacts the histones, and G/C dinucleotides in the major grooves.
  • Thanks to the lack of cleavage within linker regions, the authors were able to map nucleosome depleted regions, as well as the nearest flanking nucleosomes
    • They ranked yeast genes based on nucleosome crowding and showed that the most frequently transcribed genes have decreased nucleosome spacing and larger nucleosome depleted regions

The combination of nucleosome phasing data and accurate nucleosome mapping led the authors to develop a simple biophysical model for nucleosome positioning. This exciting work begins to dissect the complex relationship between chromatin features and nucleosome position, and the role these play in gene expression.

Searching for more? No map needed. Just click over to the full article in Genome Biology, February 2018.

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Old Fathers Pass on an Unwanted Aging DNA Methylation Profile to the Next Generation http://epigenie.com/old-fathers-pass-unwanted-aging-dna-methylation-profile-next-generation/ http://epigenie.com/old-fathers-pass-unwanted-aging-dna-methylation-profile-next-generation/#respond Thu, 01 Mar 2018 17:39:23 +0000 http://epigenie.com/?p=26916 Great looks, a well-worn leather jacket, and a wide-ranging collection of old vinyl records represent great examples of inheritances that fathers happily pass on to their grateful children. However, new findings from the lab of Dan Ehninger (German Center for Neurodegenerative Diseases, Bonn, Germany) show that older dads can also pass on an intergenerational effect that detrimentally affects healthy aging in their not so grateful offspring.

Wide-ranging studies already established that children of older fathers suffer from a higher risk of disease susceptibility (including schizophrenia and Alzheimer’s disease); however, the full impact of an older father and the mechanisms controlling detrimental inheritances remain relatively unknown. To garner fresh insight, Xie and colleagues combined epigenomic, transcriptomic, and biochemical analysis to study mouse sperm and the male offspring of old (>21 months) and young (4 months) fathers.

Here’s a brief summary of the data that the authors passed on in their new report:

  • Male mice born of older fathers display reduced lifespan and worsened aging traits when compared to mice born of younger fathers
  • Genome-wide epigenetic comparisons (via reduced representation bisulfite sequencing [RRBS]) demonstrated that sperm from older males and mice born of older fathers share differentially methylated regions in promoters of genes that regulate evolutionarily conserved longevity pathways
    • The 14 differentially-methylated promoter regions controlled genes encoding components of mTOR-related cell signaling and immune-regulatory pathways
    • Epigenetic alterations correlate with changes in the expression of genes associated with cell growth and proliferation, cancer, and organismal survival
  • A combination of Ribotag technology, RNA-sequencing, and protein analyses confirmed the over-active nature of mTOR Complex 1 (mTORC1) signaling in sperm from older fathers and mice born of older fathers
    • mTOR represents a key regulator of lifespan and influences various aspects of aging and age-related pathologies via broad effects on cell growth, proliferation, metabolism, immune functions, and proteostasis
    • Interestingly, mTOR inhibition by rapamycin treatment during normal aging inhibited the development of many aging traits observed in mice born of older fathers, although drug treatment did not alter DNA methylation levels in the short term

This research focused on a mouse model, so whether human fathers will pass on a similar unwanted inheritance remains an open topic for discussion. “What we have described here are fundamental mechanisms in a mammalian model organism. These could also be relevant for humans. However, whether this is the case and, if so, to what extent has not been tested by our study and remains unknown. In this respect, our results cannot be directly applied to humans,” says Ehninger. “However, our results provide a good reason to take a closer look at this.”

To read more about some potentially unwelcome inheritance from dad, transmit yourself over to PNAS, February 2018.

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The Single-Cell Trifecta: Nucleosome Occupancy, DNA Methylation, and Gene Expression http://epigenie.com/single-cell-trifecta-nucleosome-occupancy-dna-methylation-gene-expression/ http://epigenie.com/single-cell-trifecta-nucleosome-occupancy-dna-methylation-gene-expression/#respond Tue, 27 Feb 2018 17:50:48 +0000 http://epigenie.com/?p=26914 While recent advancements let us map the epigenome of a single-cell, the same studies can leave us a bit single-minded when facing the many layers of the epigenetic landscape. Thankfully, to quench our integrative omics thirst, a clever new technique reveals that just because you’re looking at single cells doesn’t mean you only need to consider a single solitary mark.

This new sequencing technique comes at you from the labs of Wolf Reik (Babraham Institute, UK) and Oliver Stegle (EMBL-EBI, UK). A previous collaboration between the two groups gave us single cell methylome and transcriptome sequencing (scM&T-seq), and now, the talented team throws nucleosomes occupancy into the mix by adapting Nucleosome Occupancy and Methylation sequencing (NOMe-seq) to give us Single-cell Nucleosome, Methylation, Transcription sequencing (scNMT-seq).

Here’s how scNMT-seq works:

  1. Single-cells are sorted and lysed
  2. A GpC methyltransferase labels accessible DNA
  3. RNA and DNA are physically separated
  4. RNA is subjected to library preparation and sequencing using Smart-seq2
  5. DNA is treated to library perpetration and single-cell bisulfite sequencing (scBS-seq), where CpG and GpC methylation provide information about DNA methylation and nucleosome occupancy, respectively, thanks to some clever bioinformatics

Notably, by building on NOME-seq, which uses a GpC methyltransferase to label accessible DNA, inaccessible chromatin can be distinguished from missing data, a problem that typically haunts single-cell methods. This difference provides NOMe-seq and scNMT-seq with a leg up when compared to ATAC-seq and DNase-seq.

In order to test out their fancy new technique, the group utilized mouse embryonic stem cells (mESCs) and directly compared the results to data from scM&T-seq, scBS-seq, and bulk BS-seq. Their tests established that not only does scNMT-seq reveal insight into marks in isolation, but it also exposes known coordinated events at distinct regulatory elements. Moving past comparisons to known data sets, scNMT-seq was also used to detail novel interactions at individual loci. Finally, the team journeyed down the epigenetic landscape and examined differentiating mESCs, where they discovered distinct dynamics in the coupling of all three molecular layers at developmentally relevant regions.

Co-senior author Wolf Reik shares, “It’s easy to see that different types of cells would have different genetic activity. But transcription in individual cells can also vary between cells of the same type, which isn’t quite as intuitive. As biologists we want to get to the bottom of this and understand what differences are ‘normal’, which ones might signal disease and how everything fits together. Using our new technique, we will be able to understand how changes in gene expression occur between cells of the same type, and what they could mean for the future of each cell.”

Go single in on this triple play over at Nature Communications, February 2018

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dCas9-Tet1 to the Rescue: Maintained DNA Demethylation of Fragile X Syndrome Neurons http://epigenie.com/dcas9-tet1-rescue-maintained-dna-demethylation-fragile-x-syndrome-neurons/ http://epigenie.com/dcas9-tet1-rescue-maintained-dna-demethylation-fragile-x-syndrome-neurons/#respond Mon, 26 Feb 2018 21:22:12 +0000 http://epigenie.com/?p=26911 The call for a rescue operation often makes humanity turn to its latest and greatest tools and equipment. Whether it’s firefighters with water jetpacks or cutting-edge epigenetic editing technology, threats to human kind don’t have it easy these days. In their latest report, the epigenetic emergency response team from the labs of Rudolf Jaenisch and Richard Young at MIT applied their in vitro and in vivo DNA methylation editing toolbox to rescue human neurons with Fragile X Syndrome (FXS); the leading genetic cause of intellectual disabilities in males.

FXS is caused by a CGG expansion in the 5’ UTR of the FMR1 gene, where DNA hypermethylation of the expansion represses gene expression. To reverse this repressive pattern and gain insight into the epigenetic mechanisms of FXS, the talented team invoked some precision DNA demethylation via dCas9-Tet1.

Here’s what happened:

  • Lentiviral transduction of patient-derived induced pluripotent stem cells (iPSCs) with dCas9-Tet1 and a sgRNA targeting the FMR1 repeat successfully demethylated the CGG repeats to induce gene (FMR1) and protein (FMRP) expression
  • Anti-Cas9 ChIP-BS-seq and RNA-seq analysis demonstrated minimal off-target effects, as the largest changes occur at the desired target site
    • The off-target effects can be further minimized by titrating the dCas9-Tet1 expression level
  • ChIP-seq of the edited patient-derived iPSCs revealed that the non-targeted upstream FMR1 promoter switches from a silenced heterochromatic state to an active chromatin state
    • This analysis revealed an enrichment of RNA polymerase II and active chromatin marks (H3K4me3 and H3K27Ac), but a reduction in the levels of repressive chromatin marks (H3K9me3)
  • Time course experiments revealed that:
    • Expression of FMR1 is first detected 9 days after transfection, with peak expression at 3 weeks
    • DNA demethylation and gene expression persist for at least 2 weeks after using anti-CRISPR type II-A 4 (AcrIIA4) to block binding of dCas9-Tet1 to the FMR1 target site, suggesting that constitutive expression of the dCas9-Tet1 system may not be required
  • Derivation of post-mitotic neurons from the edited FXS iPSCs revealed that editing rescued the hyperactive electrophysiological phenotype
  • Moving in vivo, the team engrafted neuronal precursor cells (NPCs) derived from the edited FXS iPSCs into post-natal day 1 mouse brains, where they observed maintained FMR1 expression for up to 3 months
  • Finally, the team demonstrated successful editing of neurons derived from FXS iPSCs

First Author Shawn Liu shares, “We showed that this disorder is reversible at the neuron level. When we removed methylation of CGG repeats in the neurons derived from fragile X syndrome iPS cells, we achieved full activation of FMR1.”

Senior Author Rudolf Jaenisch concludes, “These results are quite surprising — this work produced almost a full restoration of wild type expression levels of the FMR1 gene. Often when scientists test therapeutic interventions, they only achieve partial restoration, so these results are substantial. This work validates the approach of targeting the methylation on genes, and it will be a paradigm for scientists to follow this approach for other diseases.”

Go learn how dCas9-Tet1 can rescue your experiments over at Cell, February 2018.

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Placeholder Nucleosomes Prevent DNA Methylation to Promote Embryo Progress! http://epigenie.com/placeholder-nucleosomes-prevent-dna-methylation-promote-embryo-progress/ http://epigenie.com/placeholder-nucleosomes-prevent-dna-methylation-promote-embryo-progress/#respond Mon, 26 Feb 2018 08:16:41 +0000 http://epigenie.com/?p=26906 After coming up against an impassable obstacle, researchers often need to hold their place in one research line and open up another to explore new investigational avenues and uncover new and unexpected findings. The study of epigenetics during fertilization and the early development of human embryos remains a challenging task on multiple levels, thus making researchers hold their place in human research and turn to handy model animals such as the zebrafish (Danio rerio).

However, the mechanisms in zebrafish embryos that promote the selective maintenance of paternally inherited DNA methylation patterns (from the sperm) and reprogramming of maternal patterns (from the oocyte) remain relatively unknown.  That is, until the lab the lab of Bradley R. Cairns (Huntsman Cancer Institute, University of Utah, USA) came along with their new study in zebrafish.

In their latest report, the talented team now demonstrates that establishing unmethylated DNA sequences in the genome of sperm and the early embryo, as well as controlling the gene expression programs required for the progression of embryonic development, involves a newly discovered “Placeholder” nucleosome containing histone H2A variant (H2A.Z) and H3K4me1.

The precise particulars of this pristine new paper on Placeholder nucleosomes include:

  • Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) assessing levels of promoter-associated features (H3K4me3, H3K27me3, H3K14ac, and H2A.Z) and enhancer-associated features (H3K4me1 and H3K27ac) in zebrafish sperm and early embryos established that Placeholder nucleosomes occupy genomic regions lacking DNA methylation
    • Interestingly, these regions represent promoter sequences for genes controlling embryonic development, such as housekeeping genes and early embryonic transcription factor genes
    • Using engineered gene knock-out zebrafish, the authors discovered that the Anp32e chaperone, which controls H2A.Z occupancy in the genome, acts to prevent unwanted transcription events and ensure the precise execution of transcriptional programs
    • Additionally, Placeholder nucleosomes reprogram the maternal genome to match that of the sperm, allowing epigenetic synchronization of the embryo and developmental progress
  • Following the activation of genome-wide transcription during embryo development, Placeholder-marked regions resolve to either transcriptionally active H3K4me3-modified chromatin or a transcriptionally poised bivalent state marked by H3K4me3 and H3K27me3
    • The authors suspect that this mechanism orchestrates embryonic development via the synchronized expression or silencing of crucial genes
  • Gain-of-function/loss-of-function approaches also established that the loss of Placeholder function led to DNA methylation accumulation, whereas more widespread Placeholder function promoted the expansion of DNA hypomethylation and unwanted gene activation

But what does this new scientific progress mean in the bigger picture?  “The implications of this work include a possible mechanism for how environmental factors — for example, smoking — might influence inheritance of traits, by affecting how developmental genes are packaged. These packaging states help to define whether and how genes are expressed in normal development. Such genes, if misregulated can lead to developmental disorders, and perhaps to predisposition for cancer,” says study leader Cairns. “By better understanding what happens in normal development, it opens up new possibilities for being able to identify the precursors to diseases like cancer, when cell development goes haywire.”

So, hold your place in the paper you were pondering over, and see all the progress and peruse this unparalleled piece on Placeholder nucleosomes at Cell, January 2018!

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DNA Methylation Links Prenatal Famine Exposure to Adulthood Metabolic Conditions 6 Decades Later! http://epigenie.com/dna-methylation-links-prenatal-famine-exposure-adulthood-metabolic-conditions-6-decades-later/ http://epigenie.com/dna-methylation-links-prenatal-famine-exposure-adulthood-metabolic-conditions-6-decades-later/#respond Sun, 18 Feb 2018 19:51:34 +0000 http://epigenie.com/?p=26816 Researchers from Leiden University Medical Center in the Netherlands are back at it again: studying the long-term epigenetic effects of prenatal famine utilizing the Dutch Hunger Winter Cohort. The Dutch Hunger Winter was a famine that devastated the Netherlands in the winter of 1944-1945 amidst WWII, which produced a cohort that were in utero during this time frame, allowing for a population study of prenatal famine. Those individuals exposed to prenatal famine were found to be at a higher risk for malignant metabolic phenotypes as adults.

History lesson aside, previous work from the lab identified differentially methylated regions in adult individuals exposed to prenatal famine. Now, they have taken it one step further to more definitively identify DNA methylation as the link to adulthood metabolic conditions. Here, the talented team utilized Illumina’s 450K BeadChip array to screen over 450,000 methylation sites from whole-blood samples of 422 famine cohort members and 463 sibling controls to identify specific CpG sites associated with both prenatal famine and adult metabolic phenotypes. Mediation analysis was then used to determine the extent to which DNA methylation mediated the link between prenatal famine and adult outcome.

In their quest to find CpG sites linking prenatal famine to both BMI and serum triglyceride levels in adults, the talented team discovered that:

  • A single CpG site mediates 13.4% of the relationship between prenatal famine and adulthood BMI
    • Methylation at this CpG site is associated with PIM3 expression, which is involved in energy metabolism and glucose stimulated insulin secretion
  • Six CpG sites mediate the relationship between prenatal famine and serum triglycerides
    • Aggregately, these six sites mediate 80% of the relationship
    • Methylation at the various identified CpG sites is associated with expression of the nearby genes including TXNIP, which is associated with β cell function and insulin production
  • Focused analysis of those exposed to famine during early gestation (1-10 weeks) revealed two additional CpG sites linked to serum triglycerides, at PFKFB3 and METTL8 introns
    • These two sites account for 36.3% of the relationship between early exposure to prenatal famine and serum triglyceride levels
    • PFKFB3 and METTL8 are involved in glycolysis and adipogenesis, respectively

This work establishes DNA methylation as the link between prenatal famine and adulthood metabolic conditions. Co-senior author Bastiaan Heijmans concludes that “the nine genes we found are compelling candidates to explain the link between the prenatal environment and adult health. Now we need functional studies to unravel how an altered epigenetic regulation of these genes could impinge on metabolism in the long run.”

Although it may seem grim to consider that such adulthood phenotypes are being influenced from the earliest weeks of development, Heijmans added an optimistic note: “I hope that epigenetic studies like ours will at some point allow doctors to detect and treat health risks well before the development of disease.”

To learn more about the epigenetics involved in prenatal famine, click over to the full article in Science Advances, January 2018.

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Oh Sugar! Gestational Fructose Exposure Linked to Long-Term Brain DNA Methylation Changes http://epigenie.com/oh-sugar-gestational-fructose-exposure-linked-long-term-brain-dna-methylation-changes/ http://epigenie.com/oh-sugar-gestational-fructose-exposure-linked-long-term-brain-dna-methylation-changes/#respond Fri, 16 Feb 2018 19:02:47 +0000 http://epigenie.com/?p=26810 Here at EpiGenie, we don’t like to sugar-coat our headlines. The sickly-sweet truth is that fructose can be pretty bad for your health. Fructose has become a common additive in many foods in the form of high-fructose corn syrup. Sugary drinks are one of the most common forms. Heavy soft drink consumption is linked to heart disease, stroke, diabetes, and cognitive decline. However, the gestational effect of high fructose consumption remains unclear.

Koji Ohashi’s group at Tottori University (Japan) wanted to investigate whether gestational fructose had long-term epigenetic consequences in the brain. This group previously showed that in rats, excess maternal fructose consumption alters hippocampal gene expression in offspring. Others have shown that excess maternal fructose consumption impairs hippocampal-dependent learning and memory. However, no previous study has examined the role of epigenetics in mediating these effects. Ohashi’s group focused on the hippocampus, since it is known to be highly sensitive to prenatal/early life stress. Environmental stress and malnutrition can cause inflammation, apoptosis, and reduced levels of neurotrophic factors resulting in decreased hippocampal neurogenesis. The authors hypothesised that a maternal high fructose diet would result in altered brain-derived neurotrophic factor (Bdnf) expression & methylation, reduced neurogenesis, and impaired hippocampal-dependent learning in offspring. They provided rats with access 20% fructose or 20% glucose compared to a water control during gestation and lactation.

Here’s what they found when examining juvenile (day 21) and adult (day 60) offspring:

  • High fructose offspring spend significantly less time exploring a novel object than either other group as adults, suggesting impaired learning
  • Similarly, in a fear conditioning task, the high fructose offspring exhibit less freezing behavior in the contextual task than either other group, but there are no differences in the auditory-cued fear memory task, implying impaired hippocampal- but not amygdala-dependent learning
  • High fructose offspring (but not glucose) have 20% fewer BrdU/NeuN hippocampal neurons, while there is no significant difference in apoptosis, suggesting reduced neurogenesis
  • qPCR revealed that the BDNF gene expression was reduced in high fructose (but not glucose) juvenile offspring but only in high fructose males at adulthood
  • Pyrosequencing uncovered that the CREB binding site in the Bdnf promoter is hypermethylated in juvenile and adult male fructose-exposed offspring. A luciferase assay showed that deletion of the affected CG sites affects BDNF gene expression, suggesting a functional relationship

Taken together, the findings demonstrate that gestational fructose leads to long-term changes in the epigenetic regulation of Bdnf, which lead to decreased cognitive performance and reduced adult hippocampal neurogenesis. If high gestational fructose acts as an epigenetic disruptor of neurogenesis, it would add yet another entry to the long list of reasons to be sure as sugar to avoid it.

Catch the rest of this sugar rush over at FASEB, January 2018


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Epigenetic Aging Analysis Suggests That TERT Isn´t the Anti-aging Target You Are Looking For! http://epigenie.com/epigenetic-aging-analysis-suggests-tert-isnt-anti-aging-target-looking/ http://epigenie.com/epigenetic-aging-analysis-suggests-tert-isnt-anti-aging-target-looking/#respond Tue, 13 Feb 2018 18:11:21 +0000 http://epigenie.com/?p=26808 This isn’t some sort of Jedi mind trick, we promise, but TERT may not be the anti-aging target you are looking for! TERT, or telomerase reverse transcriptase, plays an essential role in the elongation and maintenance of telomeres and as telomeres shorten with age, many surmise that activation of TERT represents an exciting approach to stave off the signs of aging.

However, a recent study from a multidisciplinary team of padawans and apprentices led by the Jedi Masters Ken K. Ong, Kenneth Raj, Kathryn L. Lunetta, and Steve Horvath has now established that longer telomeres do not correlate to younger biological age as measured by the DNA methylation level of select CpGs on the 450K array, a measure known as the epigenetic clock. Of note, individuals exhibiting an epigenetic age older than their chronological age display a higher risk of all-cause mortality, while the children of parents who reach over 100 years of age generally exhibit a younger epigenetic age.

Here are the details of this fascinating new study:

  • The authors undertook a genome wide association study (GWAS) of white blood cells from around 10,000 individuals to discover genetic variants associated with accelerated epigenetic aging
    • 5 loci correlated to intrinsic epigenetic age acceleration (IEAA), a measure based on 353 CpGs that is independent of age-related changes in blood cell composition
    • 3 loci correlated to extrinsic epigenetic age acceleration (EEAA), a measure based on 71 CpGs that takes the contribution of blood cell composition into consideration
  • Interestingly, one loci associated with IEAA mapped to the TERT gene
    • This finding associates accelerated epigenetic aging with telomere elongation/longer telomeres, an unexpected outcome
    • In vitro analysis confirmed that TERT expression in human primary fibroblast cells leads to increased epigenetic aging, which studies have linked to multiple detrimental signs of aging
  • Mendelian randomization analyses also revealed that age of onset of first menstruation (menarche) and menopause have causal effects on IEAA

Co-author Douglas P. Kiel provides some more thoughts, “TERT is a subunit of the enzyme telomerase, which is a widely known enzyme because it has been touted as an anti-aging enzyme. It has been called a modern fountain of youth. However, some scientists have pointed out that it is unlikely to become a source of anti-aging therapies. Our study highlights the error in the notion that activation of telomerase (as advocated by some) will cure aging. Instead, our study shows that an anti-aging therapy based on telomerase expression would be accompanied by continued aging.”

For a more in-depth look for the data you are most definitely looking for, take your speeder over to Nature Communications, January 2018.

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Epigenetic Editing Misses the Mark: dCas9-DNMT3A Causes Off-Target Genome-Wide DNA Hypermethylation http://epigenie.com/epigenetic-editing-misses-mark-dcas9-dnmt3a-causes-off-target-genome-wide-dna-hypermethylation/ http://epigenie.com/epigenetic-editing-misses-mark-dcas9-dnmt3a-causes-off-target-genome-wide-dna-hypermethylation/#respond Mon, 12 Feb 2018 20:17:04 +0000 http://epigenie.com/?p=26805 While we’ve been captivated by the powerful applications of precision epigenetic editing via deactivated Cas9 (dCas9), we’ve also been mesmerized by the fact that epigenetic editing doesn’t always work as expected. Building on our need to take a step back and look at the bigger picture, new findings from the lab of Alexander Meissner at the Max Planck Institute for Molecular Genetics (Germany) demonstrate that epigenetic editing leaves a mark not only on target loci but also the whole-genome.

To understand the off-target effects specific to epigenetic editing systems, the talented team employed engineered mouse embryonic stem cells (mESCs) to track de novo methylation following epigenetic editing. This mESC model contains a double knockout for the de novo methyltransferases (DNMT3A & DNMT3B) as well as a transiently repressed maintenance methyltransferase (DNMT1). Therefore, their system is depleted of methylation but maintains de novo methylation caused by epigenetic editing, all without the interference of endogenous de novo methyltransferases or a highly methylated genome. Finally, the group utilized dCas9 fused to the catalytic domain of DNMT3A (dCas9-DNMT3A) to introduce de novo methylation.

Here’s what they discovered:

  • Whole-genome bisulfite sequencing uncovered that dCas9-DNMT3A leads to a rapid and global increase in DNA methylation levels (16% on Day 0, 28% on day 2, and 47% on day 7; 83% in wild-type)
    • This increase tends to occur at sites highly methylated in wild type mESCs and not in regions marked with H3K4me3 that are protected from methylation
  • Targeted bisulfite sequencing of the 20kb up- and down-stream of sgRNA target sites revealed that the addition of a single or multiple sgRNAs does not prevent the global increases in methylation
    • Interestingly, the target sites for some sgRNAs also exhibit increased methylation in the presence of dCas9-DNMT3A alone, demonstrating that the off-target effects can create a background signal that may appear as an on-target effect without this control
  • The introduction of the DNMT3A catalytic domain on its (not fused to dCas9) yields a similar global effect to dCas9-DNMT3A
  • Transient transfection of human somatic cell lines (human embryonic kidney [293T] and human breast adenocarcinoma [MCF7]) also generates off-target methylation

Overall, these findings provide some much-needed insight into the unique considerations of designing and interpreting epigenetic editing studies. The study also leaves us wondering whether the global off-target effects occur with designer DNA methylation systems, such as dCas9-Dnmt3a-Dnmt3L or dCas9-SunTag-DNMT3A, as well as the suite of other epigenetic effector domains that can be coupled with dCas9.

Set your sights off target over at Nature Communications, February 2018

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