EpiGenie | Epigenetics, Stem Cell, and Synthetic Biology News http://epigenie.com Scientific News, Technology, and Product Information Wed, 17 Jan 2018 00:54:53 +0000 en-US hourly 1 https://wordpress.org/?v=4.9.2 H3K4 Mono-Methylation Means More at Most Enhancers http://epigenie.com/h3k4-mono-methylation-means-enhancers/ http://epigenie.com/h3k4-mono-methylation-means-enhancers/#respond Tue, 16 Jan 2018 23:36:00 +0000 http://epigenie.com/?p=26778 People can have some pretty strange tastes. From mayonnaise on fries to syrup on spaghetti, there’s no accounting for personal preference. Proteins can have some interesting preferences too, where some regions of the genome look better than others. To get at these preferences, a recent publication from the Laboratory of Bing Ren at UC San Diego sought to find the histone modification tastes of proteins that prefer to bind enhancers.

Enhancers upregulate specific genes during development, but we’re still unclear on exactly how they work. Mono-methylation of lysine 4 on histone H3 (H3K4me1) is a dynamic modification that specifically marks both active and primed enhancers, while trimethylation (H3K4me3) marks promoters. The presence of H3K4me1 has been used to generate enhancer maps. However, the actual mechanistic role of H3K4me1 at enhancers remains unclear. H3K4me1/3 are both suspected to recruit chromatin modifiers/transcription factors, but identifying enhancer-specific proteins preferring H3K4me1 over H3K4me3 is no easy task.

With that goal in mind, the talented team sought to identify H3K4me1-associated proteins and their binding characteristics at enhancers. The group used SILAC (Stable Isotope Labeling by Amino acids in Cell culture) to label all proteins in HeLa cell nuclear extract prior to pull down with either H3K4me1- or H3K4me3-marked nucleosomes. By using heavy isotopes for one methylation state and light for the other, they were able to identify putative H3K4me1-prefering proteins with differential abundance in each pull down.. They also used various ChIP-seq and X-ray crystallography experiments in mouse embryonic stem cells (mESCs) to validate their findings.

Using these approaches, they found that:

  • A subset of proteins prefer binding to H3K4me1 over H3K4me3, including known enhancer-associated chromatin modifiers such as BAF (SWI/SNF) complex members
  • Knockout of the H3K4me1-specific methyltransferases KMT2C and KMT2D in mESCs result in dramatic reduction of H3K4me1 and several known chromatin modifiers at the same loci, but not H3K4me3
  • Catalytic inactivation of KMT2C/2D results in fewer changes in H3K4me1, but regions that do lose methylation also lose specific chromatin modifiers
  • Using a nucleosome remodeling assay, they found that H3K4me1-marked nucleosomes were more efficiently remodeled by the BAF complex
  • After solving the crystal structure of a specific BAF protein (BAF45C), they found that it may prefer H3K4me1 over H3K4me3 due to a small binding pocket that could not accommodate H3K4 tri- or di-methylation.

Overall, this work suggests that H3K4me1, but not H3K4me3, has an active role in recruiting the BAF complex and other chromatin modifiers to enhancers. This binding is likely important during development and differentiation. Follow up on the other proteins identified in this paper may provide further insight into enhancer function, which is still not well understood. So just like people, finding out a protein’s preferences can really help you get to know them.

Check out the full article at Nature Genetics, January 2018

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Single-Cell DNA Methylomes Reveal the Reprograming Secrets of Early Human Embryos http://epigenie.com/single-cell-dna-methylomes-reveal-reprograming-secrets-early-human-embryos/ http://epigenie.com/single-cell-dna-methylomes-reveal-reprograming-secrets-early-human-embryos/#respond Tue, 16 Jan 2018 19:05:24 +0000 http://epigenie.com/?p=26774 You’ve heard before that timing is everything, and this is doubly true in developmental biology. New research out of Fuchou Tang’s lab (Peking University, Beijing) singles in on the timing of methylation changes during embryonic development, and exposes the delicate epigenetic dance of global demethylation and targeted remethylation.

Tang’s lab is no stranger to embryonic epigenetics, previously reporting on the DNA methylation landscape in early embryos. Now this talented lab team made use of post-bisulfite adaptor tagging (PBAT) to perform whole-genome bisulfite sequencing of individual cells in human preimplantation embryos. Using this technique in euploid embryos, they interrogated changes in the genome-wide methylation status throughout pre-implantation development and early post-implantation. Additionally, they compared methylation patterns of the maternal and paternal genomes from the sperm and oocyte stages onward.

These ambitious authors analyzed over 6.5 Tb of sequencing data covering 10.8 million CpG sites to reveal the following:

  • In the preimplantation embryo, there are three major demethylation waves interspersed with two periods of de novo methylation
    • Extensive global demethylation occurs at 10-12 hours post-fertilization, the late zygote to two-cell stage, and the eight-cell to morula stage. While the first wave of demethylation occurs mostly at enhancer and gene body regions, the second two waves are primarily at introns and SINEs
    • Widespread de novo methylation occurs at the male pro-nuclear stage and the four-cell to eight-cell stage; these sites are enriched for families of repetitive elements (SINEs, LINEs, LTRs)
    • Sites of de novo methylation sites are often demethylated in subsequent developmental stages
  • Throughout the global methylation changes, the paternal genome is demethylated faster and more completely than the maternal genome
    • This does not appear to be related to genomic imprinting
  • Differentially methylated regions (DMRs) in oocytes are enriched at CpG islands, gene promoters, and SINEs, whereas the sperm DMRs are enriched in somatic-cell-specific enhancers and SINEs
  • DNA methylation is asymmetrically inherited during cell division, allowing blastomeres at the four-cell stage to be traced back to the parental cell

Overall, this work highlights the epigenetic intricacies in a developing embryo: the balance between demethylation of inherited parental sites and de novo methylation, as well as a unique timing component.

To see how the story develops further, check out Nature Genetics, January 2018

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YY1 Drives Enhancers and Promoters Loopy! http://epigenie.com/yy1-drives-enhancers-promoters-loopy/ http://epigenie.com/yy1-drives-enhancers-promoters-loopy/#respond Mon, 15 Jan 2018 09:01:54 +0000 http://epigenie.com/?p=26771 Cell culture hood in disarray? Favorite coffee cup missing? Internet running slow? What exactly drives you loopy in the laboratory?! Recently, the lab of Richard A. Young (Whitehead Institute for Biomedical Research, Cambridge, USA) has been driven round the bend in their quest to understand what controls the DNA looping process that brings together enhancers and promoters for gene regulation purposes. Thankfully (for them!), their new findings bring some much-needed harmony that firmly establishes the Yin Yang 1 (YY1) GLI-Kruppel zinc finger transcription factor as a driving force behind the loopy behavior of genes in mammalian cells!

Luckily, Weintraub and colleagues intended to keep us all in the loop:

  • Chromatin immunoprecipitation mass spectrometry (ChIP-MS) with antibodies for specific histone modifications (H3K27ac for active enhancers, H3K4me3 for active promoters) first identified YY1 as a potential mediator of DNA looping
    • CRISPR cell-essentiality screens and chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) confirmed a role for YY1 in DNA looping
    • YY1 is expressed in the majority of tissues and YY1 enhancer/promoter occupation occurs in all cell types examined
    • HiChIP analysis (a protein-centric chromatin conformation method) demonstrated that YY1 occupies sites of enhancer-promoter interactions
  • YY1 structurally regulates enhancer-promoter interaction by binding to hypomethylated DNA and then preferentially forming YY1 homodimers to generate DNA loops
    • YY1-mediated looping facilitates the expression of associated genes
    • The process appears similar to CTCF-mediated DNA looping, although CTCF binds to sites distal from enhancers and promoters to form larger loops involved in chromatin insulation
    • YY1 mediated enhancer-promoter loops tend to take shape within the larger CTCF-mediated loops
  • CRISPR-mediated deletion of YY1 binding sites or depletion of YY1 protein disrupted enhancer-promoter contact and associated gene expression
    • Previous reports combined with these findings establish a requirement for YY1 for embryonic and adult cell viability

Overall, the authors suggest that this newly discovered role for YY1 accounts for previously reported diverse functions (including gene expression changes in cancer) and almost certainly represents a general feature of mammalian gene control.

Have you been driven crazy by this exciting new study? Then keep yourself “in the loop” over at Cell, December 2017.

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TET Proteins: Defending Differentiation by denying de novo Methylation! http://epigenie.com/tet-proteins-defending-differentiation-denying-de-novo-methylation/ http://epigenie.com/tet-proteins-defending-differentiation-denying-de-novo-methylation/#respond Thu, 14 Dec 2017 07:49:41 +0000 http://epigenie.com/?p=26747 I don´t overeat! I’m not lazy! I’m not eating the agar plates when I´m working in the lab late, honest!

Psychologists consider denial as one of our most primitive defense mechanisms and recent studies of epigenetic defense mechanisms in embryonic stem cells (ESCs) by the labs of Olivier Elemento (Weill Cornell Medical College) and Danwei Huangfu (Sloan Kettering Institute, New York) have further strengthened this association.

This new study initially sought to discover any direct connections between the Ten-eleven translocation (TET) protein family-mediated DNA demethylation and transcriptional output at specific loci, as previous studies tended to be more global in their approach. Now, after some more targeted analysis, Verma and colleagues have established that TET proteins defend the lineage-specific differentiation of ESCs by denying DNA methylation at primed chromatin states known as bivalent domains, thus permitting ESCs to express differentiation-associated genes only when required. The TET enzymes themselves normally catalyze the first step of active DNA demethylation.

So what are the details of this new ESC-based study?

  • The team first created TET1, TET2, and TET3 triple-knockout (TKO) ESCs
    • TET knockout increased de novo DNA methylation at bivalent domains, but this did not alter gene expression
    • However, TET knockout increased DNA methylation elsewhere in the genome, prompting a general decrease in gene expression under self-renewing conditions
  • Focusing in on the PAX6 locus, a neural differentiation-associated factor, the authors observed increased DNA (cytosine-5)-methyltransferase 3B (DNMT3B) binding upon TET knockout
    • The study observed no alterations to PAX6 expression under self-renewing conditions
    • Following neural priming, DNMT3B-directed de novo DNA methylation of the PAX6 bivalent domain led to the repression of PAX6 gene expression and inhibited neural differentiation of TKO-ESCs
    • Epigenetic editing of the PAX6-associated bivalent domain by dCas9 fused to the catalytic domain of TET1 prompted DNA demethylation and improved neural differentiation

There’s no denying it; these findings firmly establish the requirement for DNA demethylation by the TET enzymes at bivalent domains to defend the expression of differentiation-associated genes in ESCs. The authors hope that future studies will delineate the various factors that influence methylation status at bivalent domains and apply this combined knowledge to predict cell-type-specific gene transcription patterns during differentiation.

Don´t deny yourself a quick glance at this new study, head to Nature Genetics, December 2017 now!

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Maternal Asthma Alters DNA Methylation at Autism Linked Genes in the Gardeners of the Developing Brain http://epigenie.com/maternal-asthma-alters-dna-methylation-autism-linked-genes-gardeners-developing-brain/ http://epigenie.com/maternal-asthma-alters-dna-methylation-autism-linked-genes-gardeners-developing-brain/#respond Mon, 11 Dec 2017 21:59:40 +0000 http://epigenie.com/?p=26742 Every good gardener appreciates that pruning represents an essential part of encouraging healthy development. This principle holds true not only on the macroscopic level but also at the microscopic level of our brain’s synaptic connections, which rely on pruning by its resident immune cells: microglia. In order to sculpt neurodevelopment, microglia rely on environmental signals, which has spurred the labs of Paul Ashwood and Janine LaSalle at the University of California, Davis, to investigate how they fit into the interplay of environment and DNA methylation in autism spectrum disorders (ASD).

First author Annie Vogel Ciernia shares, “We wanted to identify some of the mechanisms underlying the epidemiology findings that maternal allergic asthma increases the risk of having a child with ASD. You have this maternal allergic asthma event during pregnancy, and then you have these very long-lasting effects on the offspring’s behavior. We wanted to figure out what mechanisms might underlie some of these long-term effects. We looked at immune cells in brain, which were prime suspects for contributing to the long-term changes in the offspring.”

To accomplish this, the team utilized a mouse model of maternal allergic asthma and examined microglia in juvenile offspring. Here’s what transpired when they analyzed DNA methylation and gene expression via whole-genome bisulfite sequencing (WGBS) and RNA-seq:

  • A comparison of the DNA methylation and gene expression signatures with other cell-type specific data sets via principal component analysis (PCA) confirmed the purity of their microglia
  • The differentially methylated regions (DMRs) are primarily intronic and intergenic, enriched for in transcription factor binding sites related to early microglia development, and regulate genes involved in immune signaling pathways
  • The differentially expressed genes (DEGs) are related to neurodevelopment, specifically the shaping of neuronal connections, as well as the response of microglia to environmental signals
  • Notably, there is very little overlap between DMRs and DEGs, although the genes that do overlap are related to neurodevelopment and autism risk

Finally, as Vogel Ciernia summarizes, “The genes we identified that had differences in methylation and changes in expression showed an enrichment for genes that had been identified as genetic risk factors for autism, as well as genes that were differentially expressed in autism human brain samples.” Co-senior author Janine LaSalle adds, “This is an environmental model, but we’re coming back to the same genes that can be genetically mutated and cause autism in rare cases. That overlap with some of the genes was pretty striking.”

Overall, the findings leave Vogel Ciernia with the outlook that “The ultimate goal would be to identify the pathways that are impacted, which could be a therapeutic target to reverse changes and potentially improve behaviors. But we need to have a better handle on whether these changes are driving the condition or compensation to a disruption in brain development.”

Go see why these findings are nothing to sneeze at in Glia, November 2017.

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Hit-and-Run Epigenetic Editing Helps Breast Cells Evade Cycle Arrest http://epigenie.com/hit-run-epigenetic-editing-helps-breast-cells-evade-cycle-arrest/ http://epigenie.com/hit-run-epigenetic-editing-helps-breast-cells-evade-cycle-arrest/#respond Mon, 11 Dec 2017 17:28:53 +0000 http://epigenie.com/?p=26736 While a hit-and-run typically leads to an arrest, in the world of epigenetic editing it turns out to be the key to evading cell cycle arrest (senescence) and promoting the transformation of a law-abiding cell into a cancerous criminal! The hypermethylation of promoters at the wrong time and wrong place is a trademark of cancer, and the labs of Gabriella Ficz (Queen Mary University of London, UK) and Tomasz Jurkowski (University of Stuttgart, Germany) sought to interrogate this phenomenon. To aid their investigation, this crime-solving team employed the ultra-potent dCas9-Dnmt3a-Dnmt3L methyltransferase previously established the Jurkowski lab.

The group turned to primary human myoepithelial cells from the healthy breast tissue of multiple donors and analyzed DNA methylation via the EPIC array. They chose to target a panel of tumor suppressor genes CDKN2A, RASSF1, HIC1 and PTEN, given the link between promoter hypermethylation and breast cancer.

The team delivered their designer methyltransferase and 26 guide gRNAs targeting the abovementioned promoters by transient transfection, where the term hit-and-run represents the temporary presence of their designer system in the edited cells. Here’s what they found:

  • A greater than 20% increase in the methylation of the target genes
    • The only decrease in gene expression caused by their construct occurs in CDKN2A transcripts
  • The edited cells are hyper-proliferative and evade senescence
    • However, the edited cells are not immortal and eventually enter cell cycle arrest through a different telomere-dependent mechanism
  • RNA-seq revealed altered gene expression profiles related to senescence
    • This subset of genes more closely resembles unmodified early passage cells
  • By breaking up their panel of targets to investigate different regions of single genes, the team discovered that the repression of p16, a CDKN2A transcript, drove the observed alterations

First author Emily Saunderson shares, “This has been an amazing project to work on as there isn’t really a rule book yet when it comes to epigenetic editing using CRISPR so we’ve been learning as we go. I think a key factor to the success of the project has been the combination of expertise from different groups.”

Senior author Gabriella Ficz concludes, “It’s surprising that cells from several healthy individuals are so permissive to gaining this epigenetic change and that one ‘hit’ from an epigenetic editing tool is sufficient to set off this chain reaction of epigenetic inheritance and establish a cancer cell-like gene expression signature. Epigenetic fluctuations happen all the time in our cells. We know that, during ageing, our epigenome is progressively distorted – so called ‘epigenetic drift’. It will therefore be exciting to find out if this drift is responsible for initiating or accelerating ageing-associated diseases. Age is the biggest risk in cancer so our work highlights the importance of understanding the mechanism behind epigenetic drift.”

Drift on over to Nature Communications, November 2017

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eBook: Key Chromatin Players | H3K27 http://epigenie.com/ebook-key-chromatin-players-h3k27/ http://epigenie.com/ebook-key-chromatin-players-h3k27/#respond Wed, 06 Dec 2017 18:38:51 +0000 http://epigenie.com/?p=26724 Histone H3 lysine 27 (H3K27) is one of the most studied histone modifications with a complex biological role. Trimethyaltion of K27 is a hallmark of repressed transcription, while acetylation is associated with active transcription. Recent work has supported these roles as well as provided evidence for novel functions.

This ebook covers the basics of H3K27 modifications, their role in transcription, and their broader significance in disease and development. Assembled here are summaries of various notable studies that examine H3K27 modifications in five broad categories.

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One-Carbon, One Long Life: Methyl Donors Linked to Longevity http://epigenie.com/one-carbon-one-long-life-methyl-donors-linked-longevity/ http://epigenie.com/one-carbon-one-long-life-methyl-donors-linked-longevity/#respond Wed, 22 Nov 2017 18:31:23 +0000 http://epigenie.com/?p=26715 While manipulating DNA methylation has taken up most of our lifespans, the manipulation of methyl donors critical to this process may just be the key to making up for the lost time.

During one-carbon metabolism, the essential amino acid methionine is metabolized into S-adenosylmethionine (SAM), a methyl donor responsible for DNA and histone methylation. Donation of the methyl group by SAM produces S-adenosylhomocysteine (SAH), which also acts as a feedback inhibitor for DNA methylation.

SAMTOR Senses SAM Levels for the mTOR Pathway

If the quest for longevity has your senses tingling, you are not alone, since Gu et al. from the lab of David Sabatini at MIT have just demonstrated how a crucial longevity pathway senses SAM levels.

The mTOR complex 1 (mTORC1) is an environmentally responsive regulator of cellular metabolism and growth that responds to nutrients. The amino acids leucine and arginine activate this pathway; however, a role for other amino acids has yet to be fully appreciated.

By making use of human embryonic kidney (HEK-293T) cells, the talented team characterized a previously unstudied protein that putatively interacts with key players of the pathway, which they termed SAMTOR (S-adenosylmethionine sensor upstream of mTORC1). Here’s what the team discovered:

  • True to its name, SAMTOR binds SAM and interacts with members of the mTORC1 pathway
  • SAMTOR inhibits the mTORC1 pathway upon methionine starvation
  • Methionine activation of mTORC1 signaling requires the SAM binding ability of SAMTOR, thus demonstrating that SAMTOR lets a cell know when there’s enough methionine via the mTORC1 pathway

Co-first author Jose Orozco shares, “People have been trying to figure out how methionine was sensed in cells for a really long time. I think that this is the first time in mammalian cells a mechanism has been found to describe the way methionine can regulate a major signaling pathway like mTOR.” Senior author David Sabatini adds, “There are a lot of similarities between the phenotypes of methionine restriction and mTOR inhibition. The existence of this protein SAMTOR provides some tantalizing data suggesting that those phenotypes may be mechanistically connected.”

Co-first author Xin Gu concludes, “It is very interesting to consider mechanistically how methionine restriction might be associated in multiple organisms with beneficial effects, and identification of this protein provides us a potential molecular handle to further investigate this question. The nutrient-sensing pathway upstream of mTOR is a very elegant system in terms of responding to the availability of certain nutrients with specific mechanisms to regulate cell growth. The currently known sensors raise some interesting questions about why cells evolved sensing mechanisms to these specific nutrients and how cells treat these nutrients differently.”

Interestingly, since low methionine diets increase lifespan in rodents, the authors speculate that SAMTOR might play a role in these benefits and believe that it may be possible to control SAMTOR function by pharmacologically targeting its SAM binding pocket.

Metformin Manipulates Mitochondrial One-Carbon Metabolism to Increase DNA Methylation

On the subject of pharmacological targeting, metformin is a drug commonly employed to treat type 2 diabetes, which also comes with an unexpected side effect: it promotes longevity in healthy individuals too. This unexpected beneficial effect appears to be due, in part, to the targeting of a number of important metabolic pathways, including mTOR. However, a new epigenetic mechanism has emerged thanks to Cuyàs et al. from the lab Javier Menéndez at the Catalan Institute of Oncology (Catalonia, Spain).

Here’s what the team uncovered when examining non-cancerous, cancer-prone, and metastatic cancer cells:

  • Metformin promotes global DNA hypermethylation, which includes LINE-1 retrotransposons, by decreasing SAH levels and increasing SAM levels
    • This hypermethylation may help counter the hypomethylation typically observed in cancerous cells
  • Making use of a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet), the team established a critical connection between one-carbon metabolism in the mitochondria and the increase in nuclear DNA methylation
    • CRISPR/Cas9 knockout of a crucial component of mitochondrial complex I (part of the respiratory chain) blunted this effect, thus demonstrating a functional role for mitochondrial metabolism

The team concludes, “The induction of an energy crisis in the cell by inhibiting the respiratory chain of the mitochondria produces a decrease in the levels of SAH, the natural inhibitor of epigenetic writers. Simultaneously, metformin and its derivatives are also able to break the flow of methyl groups through mitochondria, which leads to the accumulation of SAM, the ink used by epigenetic writers.”

Longer Living Through Pharmacology

 Overall, these two studies offer up new mechanistic insight into how one-carbon metabolism shapes our lifespans. Furthermore, this new research also suggests that pharmacological targeting of key players in pathways that connect epigenetics and metabolism may one day lead us to the fountain of youth.

Go learn how to extend your lifespan over at Science, November 2017 and Oncogene, October 2017

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Visualizing CRISPR-Cas9 Genome Editing: Seeing is Believing! http://epigenie.com/visualizing-crispr-cas9-genome-editing-seeing-believing/ http://epigenie.com/visualizing-crispr-cas9-genome-editing-seeing-believing/#respond Wed, 22 Nov 2017 18:06:58 +0000 http://epigenie.com/?p=26708 Many studies have put their faith in CRISPR-Cas9 as a means to edit genes in human embryos, to fight HIV, and to explore epigenetic regulation. However, if you are someone who really needs to see CRISPR-Cas9 in action to believe it, an epic new study has you covered! So, cast away those doubts and qualms, and read on for news of the first direct visualization of CRISPR-Cas9 genome editing!

This breathtaking new study originates from the labs of Mikihiro Shibata (Kanazawa University, Japan),  Hiroshi Nishimasu, and Osamu Nureki (University of Tokyo, Japan) where researchers have employed high-speed atomic force microscopy (HS-AFM) to capture live action movies of CRISPR-Cas9 to delineate the exact mechanism of action for genome editing. Other related single-molecule imaging methods typically employ the detection of fluorescent-probe labels, rather than the direct visualization of the structures and dynamics of intact molecules at the nanometer scale afforded by HS-AFM.

Lights! Camera! Action! What did these astounding new movies divulge about the process of CRISPR-Cas9 genome editing?

  • Unexpectedly, Cas9 adopts a flexible modular architecture in the absence of a guide RNA (apo-Cas9)
    • However, the presence of guide RNA (Cas9-RNA) leads to the formation of a stable bi-lobed effector complex
    • This permits the interrogation of DNA target sites by three-dimensional diffusion rather than one-dimensional sliding
    • Recognition of the target site leads to the unwinding of double-stranded DNA to form a structure known as an R-loop, which consists of a RNA–DNA hybrid and the displaced non-target DNA strand
  • Real-time visualization of the Cas9-mediated DNA cleavage process demonstrates that the Cas9 HNH nuclease domain fluctuates between intermediate (I) and active docked (D) states upon DNA binding
    • Drastic structural transitions mediated by the docking of the HNH active site to the cleavage site in the target DNA (near the scissile phosphate of the target strand) lead to the formation of the catalytically-active docked conformation and cleavage of the target strand
    • Meanwhile, the RuvC nuclease domain cleaves the non-target strand

These astounding nanoscale movies of complex assembly, target search, and target cleavage are surely enough to make even the most ardent skeptic a CRISPR-Cas9 believer! As the researchers (or should we say directors?) note “this study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems.”

CRISPR-Cas9 in Action! (CC BY 4.0)

For CRISPR-Cas9 genome editing, seeing is believing; for all the details see Nature Communications, November 2017.

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dCas9 Shoots Down Microsatellite Repeat Expansion http://epigenie.com/dcas9-shoots-microsatellite-repeat-expansion/ http://epigenie.com/dcas9-shoots-microsatellite-repeat-expansion/#respond Wed, 22 Nov 2017 14:51:06 +0000 http://epigenie.com/?p=26692 Dealing with microsatellite disorders has been as difficult as shooting down actual satellites, but thanks to dCas9 its gotten a lot easier. The CRISPR/Cas9 system has proven to be much more than just a pair of scissors. For instance, CRISPR interference (CRISPRi) uses deactivated Cas9 (dCas9) to create site-specific steric hinderance to perturb gene expression. A new study from Eric Wang’s lab at the University of Florida examines several dCas9 approaches to treat repeat expansion disorders.

The talented has previously examined various approaches to treat conditions such as myotonic dystrophy, a muscle wasting disorder. Type 1 (DM1) is caused by a (CTG)n triple repeat expansion in DMPK 3’UTR, while type 2 (DM2) is caused by a (CCTG)n repeat expansion in CNBP intron 1. These expanded transcripts sequester specific proteins from their RNA targets leading to abnormal RNA splicing stability. Researchers believe that the efficiency of transcription through the expanded repeats is decreased relative to non-repetitive sequences. If this is true, further impairing the transcription of the expanded allele could effectively silence it. The authors attempted this by targeting the expanded alleles from various disorders in a repeat-length dependent manner using dCas9. This would result in premature RNA polymerase termination and nascent transcript turnover of the expanded allele, while leaving the normal allele unaffected.

To do this, the authors used dCas9 and various guide RNAs to target microsatellite repeat sequences for DM1, DM2, and other repeat disorders. They hypothesized that the dCas9 proteins bound to the repeat would be sufficient to block transcription. The team optimized this system by testing various guide RNA/PAM sequence pairs for efficacy in silencing repeats of increasing length expressed from plasmids.

Here’s what they found:

  • Using plasmids in HeLa cells, only 1 of the 4 guide RNA/PAM pairs tested reduced expression of the expanded allele, and did so in a repeat-length dependent manner. This was repeated when these repeats were incorporated into the Hela cell genome
  • Targeting of (CCTG)n repeat expansion also blocked expression of the expanded gene from a plasmid in
  • In DM1 patient-derived cell lines, dCas9 treatment rescued repeat expansion phenotypes including reducing toxic RNA foci, and rescued splicing deficits
  • Using an ex vivo muscle culture from a DM1 mouse model, dCas9 application led to a ~50% restoration of muscle function

The team found that the longer the repeat, the better the dCas9-based silencing worked. This allows the non-expanded allele to be expressed normally, a major challenge for other approaches. Going forward, systems such as this may be useful as gene therapy for repeat disorders where pathogenesis is dependent on very long repeat lengths.

Get the full message over at Molecular Cell, November 2017

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