EpiGenie | Epigenetics, Stem Cell, and Synthetic Biology News http://epigenie.com Scientific News, Technology, and Product Information Sat, 03 Oct 2015 16:07:15 +0000 en-US hourly 1 http://wordpress.org/?v=4.2.5 New Reporter System Captures DNA Methylation in Real Time http://epigenie.com/new-reporter-system-captures-dna-methylation-in-real-time/ http://epigenie.com/new-reporter-system-captures-dna-methylation-in-real-time/#respond Thu, 01 Oct 2015 17:29:51 +0000 http://epigenie.com/?p=24125 If a picture is worth a thousand words then surely a video must be worth more? So far, our knowledge about DNA methylation has been limited to a comic strip of static images at different stages. That may all be about to change with a new tool reported by the lab of Rudolf Jaenisch, which may enable DNA methylation’s first steps to be caught on film.

Their system relies on one major principle: the methylation status of CpG regions can affect that of adjacent CpG sites. So, if a reporter gene is coupled to a methylation-sensitive promoter and this construct is in turn introduced next to a CpG region of choice, in theory, the activity of the reporter gene would provide a direct readout of the methylation status of this region. Neat.

Only one problem remained: finding the right methylation-sensitive promoter. The methylation status of many methylation-sensitive promoters depends on context; i.e., if a pluripotency gene is inserted into a somatic cell, its promoter will become methylated. However, the methylation of imprinted genes, which are expressed in a parent-of-origin specific manner, is controlled in cis by adjacent sequences and is not affected by tissue context. Bingo.

In their methylation reporter system, Jaenisch’s team coupled the minimal promoter from the imprinted Snrpn gene next to a fluorescent reporter gene. They then used CRISP/Cas9-mediated gene editing in mouse embryonic stem cells (ESCs) to insert the construct into the key pluripotency genes Sox2 and miR290, which are methylated and silenced during differentiation.

As expected, the fluorescent ESCs began to lose reporter gene expression during differentiation. The team used bisulfite sequencing to confirm that this event correlated with the methylation of target loci and the Snrpn promoter. Using a similar approach, they also studied the dynamics of demethylation at the same loci during cellular reprogramming.

This technique, reported in Cell, September 2015, will enable researchers to image the dynamics of DNA methylation at single cell resolution and in real time. Let’s just hope that some genes don’t prove to be camera shy.

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Cpf1 Takes CRISPR Bigger by Going Smaller http://epigenie.com/cpf1-takes-crispr-bigger-by-going-smaller/ http://epigenie.com/cpf1-takes-crispr-bigger-by-going-smaller/#respond Tue, 29 Sep 2015 23:27:11 +0000 http://epigenie.com/?p=24107 Move over Cas9; here comes yet another CRISPR-associated player. The CRISPR pioneers in the lab of Feng Zhang at MIT continue to discover the power of CRISPR , bringing us a fancy new part for genome editing. Recently, they changed things up with a slimmer Cas9 from Staphylococcus pyogenes (spCas9) but now it seems they’ve found a new star of a whole new type.

CRISPR from Prevotella and Francisella 1 (Cpf1) is a unique CRISPR effector. In the bacterial immune system, Cpf1 doesn’t require the tracRNA, which means only the cRNA is needed. When it comes to genome editing, this means a smaller sgRNA molecule is required (~42 nt compared ~100 nt), on top of the fact that Cpf1 is smaller than spCas9. However, the new features don’t end there.

Here’s what Cpf1 has to offer to genome editing:

  • It recognizes a T-rich protospacer-adjacent motif (PAM) as opposed to the G-rich PAM of Cas9, which enables new targeting possibilities in the genome.
  • When editing, Cpf1 creates sticky ends (4-5 nt), rather than the blunt ends of Cas9, which could give Cpf1 a leg up over Cas9 when it comes to ensuring proper orientation during the tricky insertions of non-homologous end joining (NHEJ).
  • This could also be advantageous in non-dividing cell types that don’t like to do the homology-directed repair (HDR) route that is Cas9’s claim to fame.
  • Interestingly, when Cpf1 cleaves, it does so further away from PAM than Cas9, which is also further away from the target site, leaving open the potential for a second round of cleavage if the correct repair pathway doesn’t happen the first time.
  • After a bit of codon optimization, two Cpf1 orthologs (from Acidaminococcus and Lachnospiraceae) were used to efficiently target DNMT1 in human cells.

Zhang concludes that “We are committed to making the CRISPR-Cpf1 technology widely accessible. Our goal is to develop tools that can accelerate research and eventually lead to new therapeutic applications. We see much more to come, even beyond Cpf1 and Cas9, with other enzymes that may be repurposed for further genome editing advances.”

Go check out next-gen CRISPR-based genome editing in Cell, September 2015

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Sorting the Old from the New – Cellular Barriers Keeps Stem Cells Young http://epigenie.com/sorting-the-old-from-the-new-cellular-barriers-keeps-stem-cells-young/ http://epigenie.com/sorting-the-old-from-the-new-cellular-barriers-keeps-stem-cells-young/#respond Tue, 29 Sep 2015 18:12:03 +0000 http://epigenie.com/?p=24102 Alongside smartphones, coffee shops, and bars, we all also need properly functioning stem cells to survive and prosper. Indeed, the stem cell theory of aging posits that the malfunctioning of stem cells in our later years, and not all that caffeine and alcohol, is the major cause of human aging. So how do we keep our stem cells young and functional for so long and what goes wrong?

A recent study, covered here at EpiGenie, demonstrated that when a stem cell divides, the stem cell copy keeps the keeps all the new pristine cellular components while the other more differentiated cell receives the old/damaged components. The authors suggest that by sorting components, the stem cell compartment can maintain optimal functionality over time. This makes perfect sense, but what controls this sorting mechanism and what changes over time?

A new study by Sebastian Jessberger has taken inspiration from work on budding yeast, which demonstrated a role for the endoplasmic reticulum (ER) in sorting cellular components, and has now shown that in a rat model:

  • Both in vitro and in vivo, neural stem cells (NSCs) also form a “barrier” within their ER membrane.
  • This barrier mediates the uneven sorting of cellular components before cell division.
    • Stem cells receiving new cellular components are highly proliferative.
    • Daughter cells receiving old/damaged components are less proliferative and in a more differentiated state.
  • However, the study also found that the ER barrier strength reduces with age.
    • This represses sorting and produces NSCs with old cellular components and lower proliferation.
  • Lamins, the nuclear envelope component that become part of the ER during cell division, control barrier strength.
    • Expression of a mutant lamin (Progerin), which causes a premature aging syndrome, weakens the barrier in young NSCs and inhibits component sorting.

Like many of the things which make up modern life, it seems that neural stem cells need shiny new parts to function properly and a lamin-mediated ER barrier helps them to achieve this. The next question is why lamin-mediated sorting fails during aging, and furthermore, whether we can pharmacologically target and alter this mechanism to keep our stem cells forever young!

So take note, get rid of those old dog-eared papers, and move this “younger” study to the top of the pile – Science, September 2015.

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Microfluidic ChIP Makes a Few Cells Go a Long Way http://epigenie.com/microfluidic-chip-makes-a-few-cells-go-a-long-way/ http://epigenie.com/microfluidic-chip-makes-a-few-cells-go-a-long-way/#respond Fri, 11 Sep 2015 16:16:43 +0000 http://epigenie.com/?p=24014 Chromatin immunoprecipitation (or ChIP) is a handy technique to study epigenetic profiles, but only if you have enough cells. The main problem with ChIP is that it can be a “greedy” technique that uses large numbers of cells while giving back the bare minimum of DNA as a result. This is problematic for the study of genome-wide epigenetic profiles in small populations of cells, such as cancer stem cells or the cells of the developing mouse blastocyst/embryo.

To solve this problem, researchers from the laboratories of Chang Lu and Kai Tan have come up with a “little” change to the ChIP process. Their new methodology, micro­fluidic oscillatory washing–based ChIP sequencing (MOWChIP-seq), generates reproducible results with as few as 100 cells and could represent a huge “little” step forward for the field.

Their new methodology involves the following steps.

  • Fabrication of a micro­fluidic chamber of only 710 nanoliters in volume, where they placed a “packed bed” of magnetic beads conjugated to an antibody.
  • Sonicated chromatin fragments were then passed through the packed bed at a slow rate to allow the immunoprecipitation of the desired target.
  • Non-specific adsorbance was removed through short oscillatory washes created by pressure pulses.
    • This allowed collection of a DNA concentration near the theoretical maximum, with a very low background level of contamination.
  • Isolated ChIP DNA can be used for sequencing library construction without pre-amplification.
  • Recovered DNA created reproducible ChIP-seq data over a wide range of input cells
    • Results generated with 100–600 cell equivalents of chromatin either matched or surpassed other small-scale ChIP technologies using 5000 to 20,000 cell inputs.
  • Direct ChIP on 100-600 cells using an optimized sonication technique derived good quality sequencing data that enabled analysis of important genome-wide features.

The authors went on to use this micro­fluidic technology to assess global histone modifications using small numbers of hematopoietic stem and progenitor cells (HSPCs) isolated from mouse fetal liver (FL). Again, the results were reproducible and consistent with known gene expression patterns. Excitingly, they were also able to distinguish previously unknown FL HSPC-specific enhancer elements.

This new micro­fluidic technique bypasses amplification steps, indexing, and pooling steps used in other ChIP strategies, and so may represent the best current technique for small-scale ChIP experiments. So have a little look and see how far your chromatin will now go at Nature Methods, July 2015.

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Microlasers Hold Big Promise for Next Generation Cellular Analysis http://epigenie.com/microlasers-hold-big-promise-for-next-generation-cellular-analysis/ http://epigenie.com/microlasers-hold-big-promise-for-next-generation-cellular-analysis/#respond Fri, 11 Sep 2015 16:14:54 +0000 http://epigenie.com/?p=24012 Recent work by Seok Hyun Yun and Matjaž Humar has shown us that laser shows aren’t just for Pink Floyd fans. These crafty researchers incorporated specific structures into individual cells, turning them into self-contained “microlasers”.

In its simplest form, laser (or light amplification by stimulated emission of radiation) technology uses an energy source to amplify photons bouncing between reflective surfaces, creating a narrow, very strong beam of light at a very specific wavelength. In this new breakthrough, the researchers used spherical structures small enough to fit inside cells as the amplification device.

Initial experiments used intracellular oil droplets or the lipid droplets stored within the body’s fat cells as the reflective surface to amplify light. In this case, the researchers used the properties of the emitted microlaser light to assess dynamic fluctuations in intracellular pressure at a single cell level – different pressures alter droplet shape and this in turn alters the light emitted by microlasers.

Substituting solid microbeads of varying forms for the oil/lipid droplets then enabled each cell to have its own distinct microlaser. Each bead, with its own specific size and shape, can generate laser light with narrow distinct parameters which can be used to identify and label that cell. Currently, overlap from different fluorescent dyes and background interference hamper conventional cell labeling, but this new technology, in theory, could individually tag every cell in the human body – that’s a jaw-dropping trillion cells!

Labeling cells using microlasers will realistically allow us to track thousands of cells within a single experiment, but the future applications of microlasers are even more fascinating. They can be further applied to measure cell movement and reaction to forces but also may be used to control photoactivatable gene expression or the activation of light sensitive drugs in a spatiotemporal manner.

So what’s the next step? The authors now hope to harness cellular energy to create the short input pulses of light needed to make fully biological microlasers. Trip the light fantastic, and enlighten yourself with this new study in Nature Photonics, July 2015.

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CRISPR Hack Enhances Stem Cell Differentiation http://epigenie.com/crispr-hack-enhances-stem-cell-differentiation/ http://epigenie.com/crispr-hack-enhances-stem-cell-differentiation/#respond Fri, 11 Sep 2015 16:08:02 +0000 http://epigenie.com/?p=24065 From horses in the farmyard to yeast in beer production, human beings have a long history of putting nature’s gifts to work. This can also be said of CRISPR technology, which utilizes a Cas9 DNA endonuclease and a guide RNA to target and destroy specific DNA sites.

Nature had intended a role for CRISPR in bacterial immune systems, but we soon realized its wider potential and put it to different uses. In stem cell research, one of CRISPR’s biggest successes has been the correction of faulty genes in induced pluripotent stem cells (iPSCs) before their subsequent differentiation into healthy replacement cells and/or tissues.

But therein lies a problem. Differentiation of iPSCs or embryonic stem cells (ESCs) is a tricky business, and the field has been searching for a means to gain control over differentiation-associated gene expression to promote effective and efficient cell differentiation.

In a major step in the right direction, researchers from the group of Timo Otonkoski have hacked CRISPR-Cas9 to generate designer gene activation factors that function in stem cells.

Their study, reported in Stem Cell Reports, evolved in two stages:

  • Spatial control with dCas9VP192
    • The group fused a Cas9 protein lacking DNA-destruction ability (dCas9) to 12 copies of a protein with potent gene activation activity (dCas9VP192)
    • Adding a promoter-specific guide RNA to the mix generated a specific gene expression activator.
  • Temporal Control with DDdCas9VP192
    • The group next fused a destabilization domain (DD) to dCas9VP192 to allow activation of gene expression only after the addition of a stabilizing drug.
  • The group then applied this system to:
    • Force pluripotency gene expression in embryonic and primary cells
    • Aid reprogramming of somatic cells into iPSCs through the activation of OCT4 expression
    • Activate the expression of specific genes in hESCs and hiPSCs to significantly boost their differentiation potential.

This CRISPR hack has generated an effective gene expression system, which if combined with large guide RNA arrays, could effectively differentiate human stem cells into functional cell populations. The authors do note that a few tweaks to this protein hack will be required first including boosting activation efficacy, enhancing destabilization control, and reducing the protein construct size.

Read all the details on how the researchers hacked CRISPR to create this exciting new gene control method at Stem Cell Reports, September 2015.

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A Polymerase Evolved for Your Bisulfite Converted DNA http://epigenie.com/a-polymerase-evolved-for-your-bisulfite-converted-dna/ http://epigenie.com/a-polymerase-evolved-for-your-bisulfite-converted-dna/#respond Fri, 11 Sep 2015 16:05:58 +0000 http://epigenie.com/?p=24057 Bisulfite conversion can be used in so many different applications that it’s not so surprisingly referred to as the gold standard for DNA methylation analysis. But getting to the gold can create some problems for DNA. The process of converting methylated DNA (5mC) to Uracil and then finally a Thymine is harsh as it involves the application of heat and basic conditions.

These conditions lead to DNA damage and the loss of precious samples. On the flip-side, using milder conditions leads to incomplete conversion and the presence of the intermediate dhU6S. Both sides of the coin bias PCR as Taq polymerase struggles to get past the bulky modified bases of damaged DNA or the conversion intermediates.

While Taq polymerase has long been the champion of PCR, the lab of Phillip Holliger previously developed another polymerase (5D4) by directed evolution to tackle hydrophobic base analogues to enable an expanded genetic code. Now they set out to characterize whether their polymerase would perform as well on the bulky bases that can arise from varying bisulfite conversion conditions.

Here’s what went down:

  • The team created a small template containing cytosines in various combinations.
  • They then tested a library of polymerase mutants, including some created by directed evolution, on DNA from different bisulfite conversion conditions.
  • Most polymerases performed poorly, although 5D4 took the trophy on damaged DNA and DNA containing the conversion intermediate dhU6S.
  • Changing templates, 5D4 chugged right past stretches of U without exhibiting the stalling typical of Taq and thus substantially outperformed Taq in regions of high GC content.
  • 5D4 appears to be better at not only ‘reading’ converted DNA but also better at utilizing non-canonical bases as a template during polymerization.

5D4 can instantly be swapped into any existing workflows and has a lot of potential in the study of rare samples, such as certain stem cell populations and precious tissue bank samples.

The team concludes with their optimistic outlook that further directed evolution using bisulfite converted DNA as a substrate could lead to even greater efficiency and sensitivity of 5D4 on bisulfite converted DNA, as these bases were not the original target.

Learn more about getting this evolved polymerase into your workflow over at Nucleic Acids Research, August 2015

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Custom-Made Histone Modifications Add Some Bling to Chromatin http://epigenie.com/custom-made-histone-modifications-add-some-bling-to-chromatin/ http://epigenie.com/custom-made-histone-modifications-add-some-bling-to-chromatin/#respond Fri, 11 Sep 2015 13:50:25 +0000 http://epigenie.com/?p=24019 Sure, the new Apple products announced this week represented new breakthroughs and yes, you’ll be able to personalize these devices with countless modifications like cases, color and screens. The real action though, was unfolding (pun intended) in the world of chromatin analysis as a new method surfaced for introducing tags or native modifications into histones (or any other protein).

This accomplishment is now possible thanks to recent work published by Tom Muir and colleagues in Nature Chemistry.

Muir’s lab took a synthetic biology approach to introduce short tags and modifications into histone H2B using ultrafast split inteins – “naturally fractured proteins [from Cyanobacteria] that tightly associate and then rapidly catalyze protein trans-splicing (PTS)”. The approach is beautiful in that it is efficient and traceless, leaving only the desired tag or native modification in place. Muir and colleagues used H2B as a model protein to test the wonders of PTS both in vivo and in vitro. They find that:

  • They could introduce tags and fluorescent probes into H2B both in vitro and in live cells
  • Addition of tag/modification could be used to assess chromatin state (euchromatin vs. heterochromatin)
  • H2BK120 ubiquitylation (a modification that cannot be mimicked with a simple mutation) can be introduced on native chromatin
  • PTS-mediated H2BK120 ubiquitylation was functional and promoted H3K79me3

Overall, this new method is complementary to current approaches to study histones and their modifications in a native context while being limited to N- or C- termini. The authors say that in principle, any modification that can be introduced into a synthetic peptide can be introduced into a histone via PTS.

So what are you waiting for? Plug in to Nature Chemistry, May 2015 and get all the details.

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Guide: Unlocking the Value in FFPE Samples with ChIP Seq http://epigenie.com/guide-unlocking-the-value-in-ffpe-samples-with-chip-seq/ http://epigenie.com/guide-unlocking-the-value-in-ffpe-samples-with-chip-seq/#respond Tue, 08 Sep 2015 21:41:18 +0000 http://epigenie.com/?p=24036 Active Motif FFPE ChIP KitInside the world’s formalin-fixed, paraffin-embedded (FFPE) tissue repositories exists an enormous store of biological information. This valuable preservation technique allows researchers to perform histological and even genetic analysis years after sample collection. 

Although the FFPE process is a valuable preservation process, it presents challenges to researchers seeking to apply epigenetic techniques such as chromatin immunoprecipitation (ChIP) and particularly, ChIP-seq.

In this guide, we’ve accumulated key steps, tips, and tricks to help improve ChIP-seq results when working with FFPE samples.



An Overview of Unique Challenges of FFPE in ChIP Seq
How does working with FFPE samples differ then regular samples? How does the FFPE process impact the ChIP process and ChIP Sequencing? We answer these questions and others.

A Step by Step Guide on Tackling FFPE Samples with ChIP Seq
From sample recovery to data output, we outline key tips for:

  • Maximizing Chromatin Recovery from FFPE Samples
  • Chromatin Preparation
  • Chromatin Yield and Quality Control
  • Improving Sensitivity of ChIP with FFPE


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CombiGEM Takes Combinatorial miRNA Analysis to the Next Level http://epigenie.com/combigem-takes-combinatorial-mirna-analysis-to-the-next-level/ http://epigenie.com/combigem-takes-combinatorial-mirna-analysis-to-the-next-level/#respond Mon, 07 Sep 2015 22:21:53 +0000 http://epigenie.com/?p=23996 Single gene-focused research can sometimes be like the Indian story of the blind men and the elephant. The blind men, having never seen an elephant before, decide to touch one and feel it for themselves, to determine its shape. However, since they all grab different parts of the elephant, each can only describe what they feel, and none of them has the full picture of how exactly an elephant looks.

Genes, such as microRNAs, tend to operate in organized complex networks, and the ability to study them in their native states has been quite technically challenging. Current combinatorial techniques for interactive network studies are limited in resolution and complexity, and they can only perform up to pairwise interaction analysis. These techniques also require substantial time investment in analysis and experimental design.

To overcome these limitations, Alan Wong and colleagues in Timothy Lu’s lab at MIT designed and developed a scalable technique to create barcoded libraries of high-order combinations of genetic elements that can be quantified with high-throughput sequencing. Their approach comprehensively characterizes high-order genetic interactions in a high-throughput fashion via an iterative cloning system. It all begins with an insert library of barcoded DNA elements.

Next, an enzymatic digestion of the pooled insert libraries, including destination vectors, followed by a one-pot ligation step, generates the combinatorial genetic library. This combination library and its insert pool “can be combined to generate higher-order combinations with concatenated barcodes that are unique for each combination, thus enabling tracking using high-throughput sequencing”.

The technology, codenamed CombiGEM (combinatorial genetics en masse), was employed to create:

  • 1,521 two-wise high-resolution libraries and 51,770 three-wise barcoded combinations of 39 human microRNA (miRNA) precursors.
  • Additionally, the researchers identified microRNA combinations that synergistically sensitize drug-resistant cancer cells to chemotherapy or inhibit cancer cell proliferation, thus providing insights into complex miRNA networks.

The researchers hope that their technology will help reveal complex interactions between multiple genetic components, possibly leading to better diagnosis, treatment, bioengineering, and all-around understanding.

Explore how to make your own combinatorial genetic library using CombiGEM at Nature Biotechnology August, 2015

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