EpiGenie | Epigenetics, Stem Cell, and Synthetic Biology News http://epigenie.com Scientific News, Technology, and Product Information Thu, 30 Jul 2015 00:19:17 +0000 en-US hourly 1 http://wordpress.org/?v=4.2.3 Webinar: Multiplex Profiling of Epigenetic Modifications – Measuring Assay Specific & Off-target Effects in the Same Sample http://epigenie.com/webinar-multiplex-profiling-of-epigenetic-modifications-measuring-assay-specific-off-target-effects-in-the-same-sample/ http://epigenie.com/webinar-multiplex-profiling-of-epigenetic-modifications-measuring-assay-specific-off-target-effects-in-the-same-sample/#respond Wed, 29 Jul 2015 18:18:28 +0000 http://epigenie.com/?p=23715

Abstract: A variety of epigenetic modifications including post-translational modifications (PTMs) on histone tails regulate gene expression and have profound effects on cell signaling and human disease. N-terminal histone tails can have a variety of modifications, such as phosphorylation, methylation and acetylation at specific amino acid residues which are conserved throughout eukaryotes and function by altering chromatin structure and creating binding sites for chromatin readers, writers or erasers. Traditionally histone PTM levels are measured by western blot or ELISA, but these require larger amounts of material and can only look at one modification at a time.

Active Motif’s Histone H3 PTM Multiplex Assay is a high throughput bead-based ELISA assay designed for use with the MAGPIX®, Luminex® 200™ or FLEXMAP 3D® instruments to enable simultaneous interrogation of the levels of multiple histone H3 PTMs within the same sample. The assay is ideally suited for high-throughput screening and profiling of histone modification levels in limited and/or compound-treated samples. The multiplexing ability means you can simultaneously screen for on-target and off-target effects within the same sample. This Luminex technology offers several advantages over traditional methods (e.g. Western Blotting) including faster turnaround times and the ability to multiplex and utilize smaller sample amounts.

This webinar will include:
• An overview of the assay and the Luminex xMAP technology and instrumentation that is the basis of the assay
• Data comparing assay performance with traditional Western blotting

The Histone H3 PTM Multiplex Assay is the newest addition to Active Motif’s repertoire of Epigenetic Services.

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Exploiting Nucleotide Recycling to Kill Cancer http://epigenie.com/exploiting-nucleotide-recycling-to-kill-cancer/ http://epigenie.com/exploiting-nucleotide-recycling-to-kill-cancer/#respond Tue, 28 Jul 2015 22:39:26 +0000 http://epigenie.com/?p=23839 Have you ever had a doubt about what exactly you can recycle? The cell is faced with the same problem when it encounters epigenetic nucleosides. It does not really know if they can be recycled as they are or discarded.

New research by the team of Skirmantas Kriaucionis shows that although such nucleotides are normally discarded, their recycling and incorporation into DNA in certain cancers constitutes a new therapeutically-exploitable avenue for cancer research.

Cells are pretty good at recycling material ingested from neighboring dying cells. This is especially true of nucleotides, through enzymes like cytidine deaminase (CDA), which participates in the recycling of deoxycytosine. Additionally, it maintains equilibrium between deoxycytosine and deoxyuracil, which provides the sole source of thymidine for the cell.

However, epigenetically-modified cytosine creates somewhat of a recycling dilemma (a bit like your mixed plastic/cardboard sandwich box) because the incorrect positioning of epigenetic modifications into DNA could have severe consequences for the cell.

Among these epigenetically-modified bases are the newcomers, 5-hydroxymethyldeoxycytosine (5hmC) and 5-formyldeoxycytosine (5fdC). After first having found that DNA polymerases cannot distinguish between these epigenetically-modified nucleotides and unmodified nucleotides when they are transfected into replicating cells, Kriaucionis and his team set out to understand what happens to the nucleosides 5-hydroxymethyldeoxycytidine (5hmdC) and 5-formyldeoxycytidine (5fdC) during nucleotide recycling. Here’s what they found:

  • Nucleotide salvage pathway enzymes cannot produce triphosphates from epigenetically-modified nucleosides, therefore, the epigenetically-modified ones do not enter the nucleotide pool.
  • Cancer cell lines overexpressing CDA died when they were treated with 5hmdC and 5fdC.
  • CDA converts 5hmdC and 5fdC into their toxic counterparts 5hmdU (5-hydroxymethyldeoxyuridine) and 5fdU (5-formhyldeoxyuridine), which, when incorporated into DNA in the form of their respective triphosphates, can induce DNA damage and cell death.
  • This process can be exploited to kill cancer cells. In two cancer xenograft mouse models, the injection of 5hmdC or 5fdC led to tumor shrinkage and DNA damage.

Melania Zauri the main author of the study says “some cancer cells, i.e. those overexpressing CDA (such as pancreatic cancer), can be targeted therapeutically by exploiting their nucleotide salvaging activity. More studies of efficacy and safety will be needed, but this study offers a clear example of how the study of basic pathways can lead to clinically relevant answers.”

Check out the full details in Nature, July 2015.

 

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Expanding the Genetic Code with Synthetic Bases http://epigenie.com/expanding-the-genetic-code-with-synthetic-bases/ http://epigenie.com/expanding-the-genetic-code-with-synthetic-bases/#respond Mon, 27 Jul 2015 21:03:22 +0000 http://epigenie.com/?p=23798  

Life on Earth only uses 4 DNA bases (ACGT), but in principle, there’s no good reason why we couldn’t use more.  Heeding the call of “this might be possible but nature hasn’t done it yet I wonder why not?”, synthetic biologists have been tinkering with synthetic bases to see if an expanded DNA alphabet could really work.  Several synthetic base pairs have been tried, and one pair was even stably maintained in a plasmid by E. coli.

However, some of these synthetic base pairs mess with the DNA structure a bit, introducing bumps and bends.  To play well with the whole range of enzymes DNA needs to interact with, it might be best for synthetic bases to form the same double-helical structure as regular DNA.

A recent pair of papers demonstrated for the first time that Z:P base pairs form the canonical DNA structure, and that the expanded DNA sequence space can hold useful new functions.

 

Z:P Pairs Nicely with Standard DNA Structure

In the first paper, headed by Millie Georgiadis, the team crystallized DNA sequences with Z:P pairs, and found:

  1. Z:P pairs form the usual Watson-Crick structure, even with a stretch of 6 Z:Ps in a row.
  2. DNA with Z:P forms the standard double helix (actually, both the A- and B-forms of the standard double helix).
  3. Z:P does shift DNA’s major groove around a bit, which could help expand its range of functions.

 

Evolution of Z:P DNA for New Function

In the second paper, led by Liqin Zhang and Zunyi Yang, the team searched the expanded sequence space of ATGCZP DNA for new functions.  Specifically, they used PCR-based, in vitro evolution to select for 25-mer sequences that would specifically bind to liver cancer cells but not to a benign control line.  Not only did Z:P pairs survive ~200 cycles of PCR, but most of the top cancer-binding sequences contained at least one Z:P.

 

Expand your own coding potential with the paper pair of Geogiadis et al. and Zhang et al. in JACS, 2015.

 

P.S., Because we know you’re curious…

Z: stands for 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone, which, curiously, does not contain a Z, and

P: stands for 2-amino-8-(1′-β-d-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one), which, curiously, does not contain a P.

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The Power of Cooperation In Biosynthetic Coculture Systems http://epigenie.com/the-power-of-cooperation-in-biosynthetic-coculture-systems/ http://epigenie.com/the-power-of-cooperation-in-biosynthetic-coculture-systems/#respond Mon, 27 Jul 2015 20:44:51 +0000 http://epigenie.com/?p=23796 Traditionally, most bioproduction culture systems use just one strain of yeast or bacteria. Sometimes, though, you just need a little help from your friends.

That’s the idea behind the emerging field of synthetic ecology, which just achieved a major proof-of-principle success thanks to a team of MIT scientists. Cocultures have been used in specific cases before, but this was the first demonstration that a synthetic ecology can solve more general problems in biomanufacturing.

The team found a way to efficiently and effectively create the key biochemical intermediate cis-cis-muconic acid (MA), an important compound used for making numerous high-demand, bulk chemicals, with a yield of 4.7 g/L and 0.35 g/g from a glucose/xylose mixture, by far the highest ever reported.

To accomplish this feat, the team designed, optimized, and scaled-up a plug-and-play E. coli-E.coli coculture system that overcomes many limitations of single-strain biosynthetic culture systems. Typical monocultures are hampered by the difficulty of coaxing all pathway enzymes to play well together within a single cell, reduced efficiency due to metabolic stress, and secretion of pathway intermediates, which interferes with substrate utilization. In the coculture approach, an upstream cell produces and secretes a key intermediate, which a second downstream cell imports and finishes processing.

Using this approach, Zhang and colleagues found that:

  • Production of MA achieved a yield of 0.35 g/g from a glucose/xylose mixture.
  • The coculture system exhibits high capacity for accommodating variable sugar mixtures and efficiently converts them to MA.
  • By mixing the same upstream cell with a different downstream producer, the system could also produce 4-hydroxybenzoic acid (4HB), another important industrial aromatic compound.

The independence of constituent cells of the coculture system makes it possible to engineer downstream cells without negative metabolic impacts on the upstream cell. As the team concludes, “these results confirmed that an E.coliE.coli coculture system can be stably scaled up to use sugar mixtures efficiently”.

Explore the technical details of this breakthrough at PNAS, July 2015.

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Human Nuclear Transfer and iPSCs: New Strategies for the Treatment of Mitochondrial Disease http://epigenie.com/human-nuclear-transfer-and-ipscs-new-strategies-for-the-treatment-of-mitochondrial-disease/ http://epigenie.com/human-nuclear-transfer-and-ipscs-new-strategies-for-the-treatment-of-mitochondrial-disease/#respond Mon, 27 Jul 2015 19:27:05 +0000 http://epigenie.com/?p=23793 We recently brought you news of an exciting study led by Juan Carlos Izpisua Belmonte in which his group successfully eliminated mutant mitochondrial (mt)DNA in oocytes to inhibit germline transmission of currently incurable mitochondrial diseases.

Well he’s back, and this time he brought a friend.  Shoukhrat Mitalipov has teamed up with Izpisua Belmonte in a new study in Nature, in which they describe how two different reprogramming techniques can generate “healthy” mutation-corrected human pluripotent stem cells (hPSCs) from patients carrying mutant mtDNA.

It is hoped that differentiation of these mitochondrially healthy hPSCs will provide much needed cell therapies for those suffering from debilitating mitochondrial diseases.

To do this, the researchers used Sendai virus-based reprogramming to generate induced pluripotent stem cells (iPSCs), and human somatic cell nuclear transfer (SCNT) to generate pluripotent stem cells (PSCs).

  • The first strategy involved the derivation of iPSC lines from fibroblasts carrying common heteroplasmic mutations (mixture of wild type and mutant mitochondrial DNA).
    • The reprogramming process generated different clones with varying levels of mutant mtDNA (heteroplasmic mtDNA segregation)
      • This allowed the isolation of iPSC lines which contained only wild type mtDNA, and not mutant mtDNA.
  • Next, the researchers employed human SCNT to generate PSCs from fibroblasts with a homoplasmic mutation (little or no wild type mtDNA).
    • The researchers transplanted a fibroblast nuclei into a human oocyte carrying healthy mitochondria to generate hPSCs lacking any parental mutant mtDNA.
    • Despite ‘unmatched’ donor mtDNA, corrected cells displayed transcriptomic profiles similar to wild type embryo-derived PSCs.
  • While fibroblasts with mutant mtDNA showed impaired oxygen consumption and ATP production, those generated from genetically rescued hPSCs displayed normal metabolic function.

This suggests that both reprogramming techniques can provide a source of healthy, mutation-corrected pluripotent stem cells with a wild type metabolism which can generate replacement cells/tissues from those suffering from mitochondrial disease.

While the iPSC route appears the simpler of the two strategies, the mechanisms behind the spontaneous segregation of mitochondria during reprogramming still remains unexplained.

See the details on how these reprogramming technologies combined to great effect at Nature, July 2015.

 

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Stem Cell Derived Brain Orginoids Reveal a Root of Autism http://epigenie.com/stem-cell-derived-brain-orginoids-reveal-a-root-of-autism/ http://epigenie.com/stem-cell-derived-brain-orginoids-reveal-a-root-of-autism/#respond Mon, 27 Jul 2015 19:14:01 +0000 http://epigenie.com/?p=23789 Two cutting edge stem cell techniques have joined forces this week to produce a breakthrough in our understanding of the causes of Autism Spectrum Disorders (ASD). Years of study have gone into assessing if specific mutations in autism-associated genes contribute to the abnormal brain development observed in ASD with little or no luck.

Seeing this problem, a group of enterprising researchers led by Flora M. Vaccarino (Yale University, USA) chose to try something new – combining the generation of induced pluripotent stem cells (iPSCs) from ASD patients and three-dimensional (3D) brain “organoid” culture techniques.

So how did they apply this new strategy and what did they discover?

  • The group generated iPSCs from ASD patients presenting with an abnormally large head/brain (macrocephaly)
    • This is a common trait linked to the clinical severity of ASD.
    • They then differentiated these cells into well ordered “mini-brains” a few millimeters in diameter in a free-floating 3D culture.
    • No common mutations existed in these cells, but the study found a common pattern of gene dysregulation: the upregulation of cell proliferation, neuronal differentiation, and synaptic assembly genes.
  • In-depth organoid analysis then found that GABAergic neural progenitor cells proliferated at a higher rate than normally observed, leading to the overproduction of GABAergic neurons.
    • This creates an overabundance of cells and an imbalance in the ratio of the inhibitory GABAergic and the excitatory glutamatergic neurons which make up most neural circuits.
  • Transcriptional analysis linked high progenitor proliferation, overproduction of neurons, and disease severity to the overexpression of a single gene – the transcription factor FOXG1.
    • FOXG1 silencing via small interfering (si)RNA limited cell proliferation and the excessive GABAergic differentiation seen in ASD organoids.

The combination of these techniques has brought great success, with FOXG1 now representing not only a molecular signature of ASD, but also a potential drug target.

But let´s think big; iPSC-derived organoid production may improve the understanding of multiple diseases and disorders of unknown origin involving any human organ and could lead to new effective therapies.

You can read this amazing new study at Cell, July 2015.

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Webinar: Key Steps for Validating Antibodies and Optimizing the ChIP Workflow http://epigenie.com/webinar-key-steps-for-validating-antibodies-and-optimizing-the-chip-workflow/ http://epigenie.com/webinar-key-steps-for-validating-antibodies-and-optimizing-the-chip-workflow/#respond Mon, 27 Jul 2015 16:12:05 +0000 http://epigenie.com/?p=23693

Abstract: Research in the field of epigenetics has grown at a rapid pace since the discovery of the first histone acetyltransferase enzymes 18 years ago. Since then, significant advances have been made in our understanding of the basic mechanisms of epigenetics (histone acetylation, histone methylation, chromatin remodeling and DNA methylation) and the impact of epigenetic deregulation on cancer, inflammation, metabolism, and neurological diseases. This impact on disease has been underscored by the recent identification of potential oncogenic mutations and losses of epigenetic regulators such as the histone methyltransferase EZH2 and chromatin remodeling protein ARID1A. The chromatin IP (ChIP) assay is a widely used application and has provided a wealth of information regarding the localization and abundance of epigenetic marks and DNA-binding proteins across the genome in many cell types and tissues. In this webinar, Dr. Christopher Fry will present on important factors to consider when performing a ChIP assay, including use of highly validated antibodies, optimized protocols and reagents, and the advantages of using enzyme-based chromatin digestion over sonication-based chromatin fragmentation.

About the Speaker: Christopher Fry is an Associate Director of Product Development at Cell Signaling Technology, where he leads two teams, one that focuses on the development of antibodies against protein and non-protein targets involved in epigenetics, and the other that focuses the development of products for chromatin IP (ChIP).

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Salivating Insights into the DNA Methylation Connection in Fetal Alcohol Syndrome http://epigenie.com/salivating-insights-into-the-dna-methylation-connection-in-fetal-alcohol-syndrome/ http://epigenie.com/salivating-insights-into-the-dna-methylation-connection-in-fetal-alcohol-syndrome/#respond Mon, 20 Jul 2015 19:27:12 +0000 http://epigenie.com/?p=23762 Things often show up where you least expect. Whether it’s finding your keys in the freezer, your phone in the couch, English kings underneath a car park, or the secrets of brain development in spit. Sometimes all you have to do is look.

Previously, the lab of Shiva Singh from the University of Western Ontario developed extensive mouse models of prenatal alcohol exposure that showed that moderate alcohol exposure causes long lasting and brain-specific changes in DNA methylation and ncRNA. In this report, the team examined Buccal swabs from young children with fetal alcohol spectrum disorders (FASD) and found some identical changes to what they had seen in adult mouse brains.

Here’s what they found using human methylation 450K arrays:

  • The swabs of FASD children clustered separately from those of carefully matched controls to reveal hundreds of informative CpGs.
  • Strangely, the alterations to DNA methylation occurred in genes, functions (ontologies), and pathways related to the brain’s synaptic signaling (glutamatergic synapses) as well as developmental (hippo signaling) and calcium signaling pathways.
  • Taking inspiration from the skyscrapers of Dubai, the clustered protocadherins gave a strong signal in the Manhattan plot. This complex locus relies on DNA methylation and CTCF to create alternative transcripts with diversity paralleling that of the immune system and generates individual neuronal identity.
  • These findings were replicated via pyrosequencing for select CpGs and verified at the omic level in a separate and more diverse replication cohort.
  • Interestingly, buccal epithelial cells and neurons of the adult brain are derived from a common precursor whose potency shifts between epithelial and neuronal state during development. Lead author Ben Laufer says that “these findings highlight the informative nature of buccal swabs for neuroepigenomics and also provide an example of mouse models being efficiently translated to human discovery.”

Laufer also shares that EpigenDx performed the gene-specific DNA methylation pyrosequencing. When examining methylation on the 450K array, many probes are often discarded for containing SNPs, which can alter their performance.

However, pyrosequencing enabled the team to maximize the value of the 450K array by not having to discard these probes because they were now able to examine for the presence of SNPs. “This was critical as some of the most informative probes were known to have CpG SNPs or SNPs in them/nearby” adds Laufer. EpigenDx has a rapidly growing database of over 4000 validated assays for human, mouse, and rat gene loci and extensive custom design experience.

They also have the track record to prove it, with a library of 60 publications using their DNA methylation services that also contains many familiar names.

Go learn about the brain from spit in Epigenomics, October 2015 and check out what EpigenDx can do for you.

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Race, Gender and Non-Coding RNA: tRNAs Don’t Play it Politically Correct http://epigenie.com/race-gender-and-non-coding-rna-trnas-dont-play-it-politically-correct/ http://epigenie.com/race-gender-and-non-coding-rna-trnas-dont-play-it-politically-correct/#respond Fri, 17 Jul 2015 19:40:23 +0000 http://epigenie.com/?p=23719 Human genetics just got a whole lot more complicated with a report published last week by Isidore Rigoutsos’ team in Oncotarget. As if differences in expression profiles between cells, tissues and differentiation stages weren’t already enough, it seems that race and gender also affect genomic output.

Specifically, the team focused on transfer RNAs (tRNAs). tRNAs may seem like unassuming molecules to some, with a dull factory-floor job in the production line of proteins. However, they can give rise to shorter tRNA fragments (tRFs) through cleavage, and while it is still unclear what these molecules actually do, they have been shown to affect many physiological processes, including cell growth and response to DNA damage.

They have been found in all corners of life, from bacteria and archaea to yeast and mammals, but they have not yet been extensively catalogued in humans.

To study the diversity of tRFs in human populations, Rigoutsos’ team focused on RNA-seq datasets from two large samples: lymphoblastoid cell lines derived from 452 healthy men and women representing five different races, and 311 samples of primary breast cancer tissue.

After some pretty impressive number crunching and displays of technical prowess required to cope with these repetitive sequences, here’s what they found:

 

  • They discovered a new type of tRF which they called ‘internal tRNA fragments’ (i-tRFs) because i-tRFs begin and end in the interior of a mature tRNA.
  • Mitochondrial tRNAs are a rich source of tRFs.
  • Characteristics of tRFs such as their abundance and length depended on disease status, race, population and gender; for example, levels of 93 tRFs differed significantly between white and black populations.

 

These findings tie in with a previous study by the same team showing that miRNA isoforms also depend on gender, population and race. Rigoutsos sums up the implications of this work. “We now have to take into account race-dependencies and gender-dependencies when studying the molecular biology of disease. Will what we learn from a cell line from someone in North Europe still apply to someone from South Europe?”

“Yes there is complexity but knowledge of that complexity means more power for researchers going forward”.

 

Check out just how complicated things can get at Oncotarget, July 2015.

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Resistance is Futile: Epigenetic Therapy Makes Cancer Cells Defenseless http://epigenie.com/resistance-is-futile-epigenetic-therapy-makes-cancer-cells-defenseless/ http://epigenie.com/resistance-is-futile-epigenetic-therapy-makes-cancer-cells-defenseless/#respond Thu, 09 Jul 2015 17:07:56 +0000 http://epigenie.com/?p=23707 A team of American scientists have shown that an epigenetic therapy might hold promise for combatting drug resistance in cancer.

Cancer cells are masters of drug evasion and adept at using alternative pathways to give them an edge during treatment, complicating many therapeutic approaches. Overcoming drug resistance in cancer cells, would save millions of lives and drastically cut treatment costs.

Epigenetic reprogramming fueled by DNA methylation changes, chromatin reconfiguration, histone modification, and/or noncoding RNAs, is very common in most human cancers. Important tumor suppressors are often epigenetically silenced either via DNA methylation or histone deacetylation and thus are unable to perform their tumor suppressive functions. As a result, reprogramming the reprogramming might help cancer treatment in resistance scenarios.

Hasanali and colleagues set out to determine whether combinatorial epigenetic therapy can provide an alternative cure for patients with T cell prolymphocytic leukemia (T-PLL). To achieve their aim, they threw the “kitchen sink” combination of demethylating agents, histone deacetylase (HDAC) inhibitors, and the immunotherapeutic agent, Brentuximab vedotin, to treat eight patients with T-PLL resistant to the conventional treatment, alemtuzumab.

 

Here is what the team learned:

 

  • Introducing epigenetic agents such as cladribine induced the expression of tumor necrosis factor receptor superfamily member 8, TNFRSF8 (CD30), a well studied and frequently targeted cancer marker
  • Adding epigenetic agents to alemtuzumab treatment overcomes resistance to alemtuzumab in T-PLL patients.
  • Remarkably, seven out of the eight patients that received this treatment achieved complete remission, and the eighth patient achieved partial remission.

 

These results add to previous data that suggest a promising future for combinatorial epigenetic therapy in cancer treatment.

 

Explore how to make cancer cells defenseless at Science Translational Medicine, June 2015.

 

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