Leafing through the Literature

Thalyana Smith-Vikos

Highlighting recently published articles in molecular biology, genetics, and other hot topics

Can I get some of your gut bacteria?

While there have been many reports popping up in the literature that demonstrate a connection between gut microbiome and diet, Ridaura et al. have elegantly showed how the mammalian microbiome affects diet in a specific yet alterable manner that can be transmitted across individuals. The researchers transplanted fecal microbiota from adult murine female twins (one obsess, one lean) into mice fed diets of varying levels of saturated fats, fruits and vegetables. Body and fat mass did depend on fecal bacterial composition. Strikingly, mice that had been given the obese twin’s microbiota did not develop an increase in body mass or obesity-related phenotypes when situated next to mice that had been given the lean twin’s microbiota. The researchers saw that, for certain diets, there was a transmission of specific bacteria from the lean mouse to the obese mouse’s microbiota.

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In vivo reprogramming

Abad et al. have performed reprogramming of adult cells into induced pluripotent stem cells (iPSCs) in vivo. By activating the transcription factor cocktail of Oct4, Sox2, Klf4 and c-Myc in mice, the researchers observed teratomas forming in multiple organs, and the pluripotency marker NANOG was expressed in the stomach, intestine, pancreas and kidney. Hematopoietic cells were also de-differentiated via bone marrow transplantation. Additionally, the iPSCs generated in vivo were more similar to embryonic stem cells than in vitro iPSCs by comparing transcriptomes. The authors also report that in vivo iPSCs display totipotency features.

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Connection between pluripotency and embryonic development

Lee and colleagues have discovered that some of the same pluripotency factors (Nanog, Oct4/Pou5f1 and SoxB1) are also required for the transition from maternal to zygotic gene activation in early development. Using zebrafish as a model, the authors identified several hundred genes that are activated during this transition period, which is required for gastrulation and removal of maternal mRNAs in the zebrafish embryo. In fact, nanogsox19b and pou5f1 were the top translated transcription factors prior to this transition, and a triple knockdown prevented embryonic development, as well as the activation of many zygotic genes. One of the genes that failed to activate was miR-430, which the authors have previously shown is required for the maternal to zygotic transition. Thus, Nanog, Oct4 and SoxB1 induce the maternal to zygotic transition by activating miR-430.

 

A microRNA promotes sugar stability

Pederson and colleagues report that a C. elegans microRNA, miR-79, targets two factors critical for proteoglycan biosynthesis, namely a chondroitin synthesis and a uridine 5′-diphosphate-sugar transporter. Loss-of-function mir-79 mutants display neurodevelopmental abnormalities due to altered expression of these biosynthesis factors. The researchers discovered that this dysregulation of the two miR-79 targets leads to a disruption of neuronal migration through the glypican pathway, identifying the crucial impact of this conserved microRNA on proteoglycan homeostasis.

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Establishing heterochromatin in Drosophila

It is known that RNAi and heterochromatin factor HP1 are required for organizing heterochromatin structures and silencing transposons in S. pombe. Gu and Elgin built on this information by studying loss of function mutants and shRNA lines of genes of interest in an animal model, Drosophila, during early and late development. The Piwi protein (involved in piRNA function) appeared to only be required in early embryonic stages for silencing chromatin in somatic cells.  Loss of Piwi leads to decreased HP1a, and the authors concluded that Piwi targets HP1a when heterochromatin structures are first established, but this targeting does not continue in later cell divisions. However, HP1a was required for primary assembly of heterochromatin structures and maintenance during subsequent cell divisions.

 

The glutamate receptor has a role in Alzheimer’s

Um and colleagues conducted a screen of transmembrane postsynaptic density proteins that might be able to couple amyloid-β oligomers (Aβo) bound by cellular prion protein (PrPC) with Fyn kinase, which disrupts synapses and triggers Alzheimer’s when activated by Aβo-PrPC . The researchers found that only the metabotropic glutamate receptor, mGluR5, allowed Aβo-PrPC  to activate intracellular Fyn. They further showed a physical interaction between PrPC and mGluR5, and that Fyn is found in complex with mGluR5. In Xenopus oocytes and neurons, Aβo-PrPC caused an increase in intracellular calcium dependent on mGluR5. Further, the Aβo-PrPC-mGluR5 complex resulted in dendritic spine loss. As a possible therapeutic, an mGluR5 antagonist given to a mouse model of inherited Alzheimer’s reversed the loss in synapse density and recovered learning and memory loss.

 

Keep playing those video games!

Anguera et al. investigated whether multitasking abilities can be improved in aging individuals, as these skills have become increasingly necessary in today’s world. The scientists developed a video game called NeuroRacer to test multitasking performance on individuals aged 20 to 79, and they observed that there is an initial decline in this ability with age. However, by playing a version of NeuroRacer in a multitasking training mode, individuals aged 60-85 achieved levels higher than that of 20-year-olds who had not used the training mode, and these successes persisted over the course of 6 months. This training in older adults improved cognitive control, attention and memory, and the enhancement in multitasking was still apparent 6 months later. The results from playing this video game indicate that the cognitive control system in the brains of aging individuals can be improved with simple training.

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Domesticated HIV Leads to Gene Therapy Success

Alisa Moskaleva

For as long as biomedical scientists have known that DNA mutations cause disease, they wanted to be able to correct them. Dozens of rare but devastating human diseases are caused by a mutation in a single gene. If a mutant form of the gene causes the disease, then giving the patient the correct form of the gene should cure it. This idea is called gene therapy. Now, two papers in the 23 August 2013 issue of Science by Dr. Luigi Naldini and co-workers report prolongation and improvement of quality of life from gene therapy for six children with rare genetic diseases. These papers are not the first to report health benefit from gene therapy and not the first to use the technique of lentivirus-mediated hematopoietic stem cell gene therapy, which involves domesticated HIV and which I’ll explain shortly. However, the papers address two previously incurable diseases, metachromatic leukodystrophy in one paper and Wiskott-Aldrich syndrome in the other, and add to the evidence that the gene-therapy technique that involves something as scary as HIV is safe.

So, what is lentivirus-mediated hematopoietic stem cell gene therapy and what does it have to do with HIV? Let’s start with “lentivirus-mediated.” Lentivirus is a virus with an RNA genome that infects eukaryotic cells. To persist in cells and reproduce, lentivirus integrates its RNA genome by converting it into DNA and adding this DNA into the genome of the host. HIV is a lentivirus that infects T-cells. By replacing the gene in the HIV genome that targets it to T-cells with a gene that recognizes all mammalian cells and further adding a gene of interest, it’s possible to use HIV to integrate the gene of interest into the genome of any mammalian cell. So, if only HIV could be made to stop replicating uncontrollably, gene therapy could use it to deliver correct forms of disease-causing genes to patients’ cells. Over the past twenty years, scientists have domesticated HIV by removing most of its genes, and constructing versions that can infect but can’t replicate. HIV-based lentivirus is now routinely used in research to introduce DNA into mammalian cells and it can be a method of gene therapy. Hematopoietic stem cell gene therapy means that hematopoietic stem cells, the cells that can give rise to red and white blood cells, are the targets of gene therapy.

Why did Dr. Naldini and colleagues target hematopoietic stem cells? Wiskott-Aldrich syndrome is a disease of white blood cells, so it makes sense to deliver the correct form of the disease-causing gene to their source. Metachromatic leukodystrophy is a disease of the brain and the peripheral nervous system, so at first glance brain cells and nerve cells should have been the targets of gene therapy. However, getting the gene to integrate in enough cells requires literally bathing the cells in a high concentration of lentivirus. It’s impossible to achieve a high enough concentration of lentivirus for cells inside the body. Delivering genes to cells inside the body remains one of the biggest challenges in gene therapy. Hematopoietic stem cells are a much easier target because they can be isolated from the body by drawing blood and sorting it. The isolated cells can then be concentrated and bathed in lentivirus before being re-injected into the patient’s bloodstream. In metachromatic leukodystrophy, the disease-causing form of the gene causes the accumulation of a toxin and the correct form destroys it. The authors hoped that hematopoietic stem cells with the correct form of the gene would give rise to cells of the immune system that would migrate to the brain and the peripheral nervous system and scavenge the toxin. Therefore, hematopoietic stem cells are a good target for gene therapy not only because they are easy to isolate but also because they can migrate and help out other cell types that cannot be targeted directly.

However, hematopoietic stem cells have a dark side. When they are injected into the patient’s bloodstream to exert their beneficial effect, they can transform into cancerous cells and cause leukemia. Leukemia was an unfortunate side effect of gene therapy against the Wiskott-Aldrich syndrome and two other rare diseases using a different method of gene delivery called gamma retrovirus that preceded lentivirus. Gamma retrovirus is also a virus that infects eukaryotic cells and integrates its genome into the host genome. Analysis of the genome of cancerous cells revealed that when the gamma retrovirus integrated, a part of its DNA ended up near a cancer-causing gene and, acting like a switch, turned it on. Mindful of this, Dr. Naldini and colleagues analyzed the genome of hematopoietic stem cells from their patients several times after treatment. Reassuringly, they did not find any cells with inappropriately activated cancer-causing genes 1.5 to 2 years after treatment in patients battling metachromatic leukodystrophy and 1.5 to 2.5 years after treatment in patients with Wiskott-Aldrich syndrome. Dr. Naldini and colleagues hypothesize that HIV-based lentivirus may be safer than gamma retrovirus because it has fewer DNA switches that can turn a gene on, and because lentivirus integrates more randomly in the genome than gamma retrovirus. They will be following their patients for years to come.

Is gene therapy with domesticated HIV safe? Only time and more research will tell. But six children who were predicted to die months ago are walking and running, playing and talking. They got a respite from dreadful symptoms, like eczema, internal bleeding, and inability to walk or even to hold up their own head. One fervently hopes for more gene-therapy good news.

DNA Damage, Cell Division, and Human-like Mushrooms

Alisa Moskaleva

Biologists often use yeast, fruit flies, mice and other so-called model organisms to understand humans, but how about using humans to understand mushrooms? In a recent paper, Carmen de Sena-Tomás and colleagues show that when it comes to the recognition of DNA damage, mushrooms are quite human-like. In addition to underscoring the relatedness of all living things, their discovery may help biologists understand how all eukaryotes control cell division.

Humans served as a model organism here because as part of the Human Genome Project in June 1995 a team led by Yosef Shiloh discovered the genetic cause of ataxia telangiectasia. This rare childhood disease is characterized by uncoordinated movement, weakened immune system, and, importantly, increased predisposition to cancer. The team named the gene ATM for “ataxia telangiectasia mutated.” A year later, Karlene Cimprich and colleagues published the discovery of a related gene that goes by the name ATR for “ataxia telangiectasia mutated and Rad3 related”. Mutations in ATR cause Seckel syndrome, an extremely rare disease that among its many symptoms also has predisposition to cancer. Cancer arises more frequently in patients with ataxia telangiectasia and Seckel syndrome because they suffer from more mutations to tumor suppressors and oncogenes as a result of DNA damage that goes unrecognized and unrepaired. The protein products of ATM and ATR genes recognize DNA damage and also prevent mitosis in the presence of high levels of damaged DNA, which can be lethal or, if the daughter cells survive, can produce still more carcinogenic mutations.

If physicians hadn’t documented these human diseases and geneticists hadn’t gone looking for the causative genes, it is doubtful whether biologists would have ever stumbled upon what is now known as the DNA damage checkpoint, consisting of ATM, ATR, and downstream proteins. Since the discovery in humans, the DNA damage checkpoint has been found in many organisms including yeast and mice. Carmen de Sena-Tomás and colleagues decided to look at Coprinopsis cinerea, a model system of mushrooms.

At first glance, mushrooms and humans are nothing alike. Though both are multicellular eukaryotes with differentiated tissues, mushrooms are threadlike scavengers that crisscross the soil with their networks of hyphae, feed by releasing digestive enzymes into their surroundings, sprout the recognizable stalk-and-cap fruiting bodies, and practice a curious version of mitosis. A mushroom cell has two haploid nuclei that do not fuse, replicate separately, and divide synchronously, so that each daughter cell once again has two haploid nuclei. Carmen de Sena-Tomás and colleagues wondered if the DNA damage checkpoint known to regulate human mitosis could be regulating mushroom mitosis as well.

They found orthologs of ATR and a target of ATR called Chk1 in the recently sequenced Coprinopsis cinerea genome. When they perturbed the function of the mushroom ATR and Chk1, they found increased sensitivity to DNA damage and increased number of abnormal mitoses where the two haploid nuclei would divide at different times and daughter cells would receive more or fewer than two nuclei. These results lead to two important conclusions. First, ATR and Chk1 in mushrooms recognize DNA damage, just like in humans and many other organisms. Second, ATR and Chk1 prevent abnormal mitosis.

Carmen de Sena-Tomás and colleagues speculate that ATR and Chk1 prevent abnormal mitosis by sensing whether or not the mushroom cell has completed DNA replication and repair. Since the DNA damage checkpoint is so similar in mushrooms and humans, it is tempting to propose that all eukaryotes use ATM and ATR to prevent mitosis before the completion of DNA replication and repair. Though tempting, this is not established. Carmen de Sena-Tomás and colleagues have no direct evidence. The most direct evidence comes from experiments with purified ATR and damaged DNA, which may or may not accurately represent the situation inside living cells. Moreover, there is opposing evidence from yeast that the DNA damage checkpoint does not react to the low levels of DNA damage present in most cells before mitosis. And further evidence points at proteins distinct from ATM and ATR and collectively known as the DNA damage tolerance pathway as the guardians of mitosis from DNA damage. That’s why the effect of the DNA damage checkpoint on mitosis in mushrooms discovered by Carmen de Sena-Tomás and colleagues is important. It doesn’t provide a complete answer about what happens in all eukaryotes, but it should reinvigorate the debate.

Shining Light on Microbial Dark Matter

Sophia David

Microorganisms are the most abundant and diverse cellular life forms on the planet. Unfortunately though, we have only been able to culture a small subset of microbial species in the laboratory. These represent just a tiny fraction of the environmental diversity. Furthermore, we have only been able to sequence the genomes of organisms that we can culture. Our knowledge of microorganisms is therefore highly biased towards cultivated bacteria and archaea that almost certainly do not represent the full environmental diversity.

Research published two weeks ago in Nature by a team led by Tanja Woyke from the DOE Joint Genome Institute in California has attempted to address this issue. The researchers used an emerging technique Continue reading “Shining Light on Microbial Dark Matter”

Mysteries of Aneuploidy

Nicole Crown

I’ve often wondered why humans are so bad at reproduction.  It’s been estimated that 10-30% of all fertilized eggs are aneuploid, and approximately one third of all miscarriages are due to aneuploidy (Hassold et al.).  In striking contrast, only 1-2% of fertilized eggs are aneuploid in mice.  As a researcher that studies meiosis, this baffles me.  The meiotic program is astoundingly complex, coordinating the repair of programmed DNA damage, finding and pairing homologous chromosomes, along with attaching the spindle in the correct orientation.  This complexity requires multiple checkpoints throughout the process to ensure everything is going as planned; if something goes wrong, the checkpoint stops the cell. Given multiple opportunities to ensure chromosomes are properly segregated, how is it that, in humans, aneuploid cells not only make it through a complete meiosis, but also go on to complete oogenesis?

Recent work from Dokshin et al. suggests that one potential way this can happen is to decouple oogenesis from meiosis.  In mice, early germ line cells initiate meiosis after Stra8 expression is turned on by a retinoic acid signal.  In the absence of Stra8, cells never initiate meiosis, and by 6-8 weeks of age, the ovaries contain no germ cells. However, a very small percentage of cells are able to escape the Stra8 phenotype, and while they still don’t initiate meiosis, they do proceed through oogenesis.  These “oocyte-like cells”, as the authors call them, have the same morphological and physiological characteristics as normal oocytes: they are able to make a zona pellucida, generate follicles, can be ovulated and fertilized, and the embryo can undergo the first division.  When the authors looked at the chromosome complement of the oocyte-like cells, they found that the chromosomes were randomly distributed between the polar body and the oocyte-like cell.

Certainly, these data show that in the absence of meiosis, the vast majority of female germ cells will not differentiate; therefore, there must be some way of monitoring the meiotic state of a cell before oocyte differentiation begins.  However, this monitoring clearly can fail, as the Stra8 deficient mice do produce oocyte-like cells capable of being fertilized.  The authors suggest some cases of human infertility may be explained by a disconnect between oocyte differentiation and meiosis.  It will be fascinating to compare the mechanism of communication between meiotic state and oocyte differentiation in humans to other organisms, and determine if the apparently higher rate of aneuploidy in humans is sometimes due to miscommunication.

Stick a PIN in it

Nicole Crown

Post-translational modification (PTM) of proteins is an essential cellular process used to regulate protein function and stability.  PTMs are assumed to have an impact on protein structure and conformation, but the effects of specific protein conformations on biological processes are difficult to understand and test in vivo.  The importance of protein conformation is of particular interest in DNA repair as many of the same proteins are involved in context specific repair processes.  For example, the Mre11-Rad50-Nbs1 complex is an essential DNA double-strand break sensor that is found at every type of double-strand break.  It has been proposed that this complex can, in theory, adopt up to 216 conformational states and acts as a “molecular computer” to detect damage and regulate repair pathway choice (i.e. should the break be repaired via nonhomologous end joining or homologous recombination)1In vivo studies of the roles that particular protein conformations play are rare, yet critical for understanding the regulation of DSB repair.

New work from Steger and colleagues2 shows that Pin1, a prolyl isomerase that catalyzes cis/trans isomerization, binds multiple DNA repair proteins, including CtIP, a key player in double-strand break end resection.  The authors show that the interaction between Pin1 and CtIP is induced by phosphorylation of CtIP at two S/T-Proline sites, and that Pin1 binding does indeed cause a conformational change in CtIP.  This conformational change leads to polyubiquitylation of CtIP and subsequent degradation. Overexpression of Pin1 causes hyporesection and a decrease in homologous recombination; similarly, depleting Pin1 causes hyperresection and decreased NHEJ.  These data and others presented in the paper lead to a model in which CtIP is phosphorylated, causing Pin1 to bind and isomerize CtIP.  This isomerization leads to ubiquitin-mediated CtIP degradation and appropriate end resection.  Pin1 overexpression leads to reduced CtIP activity and therefore hyporesection and decreased homologous recombination, whereas Pin1 depletion leads to increased CtIP activity, hyperresection and decreased NHEJ.

In this particular case, the researchers were lucky to find a specific protein, Pin1, that induces a known conformational change, cis/trans isomerization, by acting directly on the substrate protein. However, most conformational changes may be caused indirectly by the effects of post-translational modifications.  While the identification of Pin1 as a critical regulator of DNA repair is a large step forward in understanding the role protein conformation plays in function, this same understanding for other proteins will require more cross-disciplinary studies that are able to modify protein conformation in vivo and determine the biological outcomes.

1. Williams, G. J., et al. (2010). “Mre11-Rad50-Nbs1 conformations and the control of sensing, signaling, and effector responses at DNA double-strand breaks.” DNA Repair 9(12): 1299-1306.

2. Steger, M., et al. (2013). “Prolyl Isomerase PIN1 Regulates DNA Double-Strand Break Repair by Counteracting DNA End Resection.” Molecular Cell 50(3): 333-343.