the biofixer

Transcription Factor in Glioma Cells Regulated by Novel Genes

Scientists discovered a cellular pathway in the deadly brain cancer malignant glioma, a pathway essential to the cancer’s ability to grow—and a potential target for therapy that would stop the cancer’s ability to thrive. 

       A genome-wide RNAi screening tool was used to identify a dozen genes that affect the function of a crucial protein necessary for glioma cells to grow. In addition, the key pathway appears in laboratory cultures and mouse models to be susceptible to two cancer drugs already in use for other types of cancer.

      A hallmark of cancer is uncontrolled cell growth, often caused by over-expression of genes that help cells survive, or under-expression of those genes that induce normal cell death. Genes that are expressed highly in cancer cells and are essential for their survival are attractive targets for drug therapy.

      Recent research revealed the essential cellular survival pathway, CREB3L2-ATF5-MCL1, in malignant glioma. The team identified novel genes that regulate the expression of a transcription factor called ATF5 (activating transcription factor 5) in malignant glioma cells. 

      ATF5 is linked to many cellular function including cell cycle progression, metabolite homeostasis cellular differentiation and apoptosis. ATF5 is a member of basic-region leucine zipper (bZIP) proteins family which binds the cAMP response element (CRE) consensus sequence: 5’GTGACGT(C/A)(G/A). This sequence is present in many viral and cellular promoters. ATF within or between subgroups can form homo- or hetero-dimer through the bZIP domain and the dimer can then bind to the DNA through the basic-motif and function as a transcription factor. Recently, another novel ATF5 consensus DNA binding sequence (CYTCTYCCTTW) was found in C6 glioma and MCF7 using a cyclic amplification and selection of targets.
      The discovery of at least one previously unknown genetic pathway that appears to regulate this key transcription factor, and the subsequent determination that the cancer drugs sorafenib and temozolomide inhibit glioma growth, indicate new possibilities for potential therapeutics.

(Source: Biotechdaily )

Decisive Role in Cell Division Identified for Centrosomal Protein

The answer to an elusive question about signaling in chromosome distribution and separation has been provided by the discovery of a key role for a centrosomal protein kinase. The kinase also has potential importance as a new candidate among cell division factors being targeted in the development of drug treatments for cancer.

      The protein kinase Nek9 has been highlighted as an essential and decisive factor in a pathway involved in ensuring efficient and accurate movement of chromosomes during cell division. γ-tubulin recruitment to and accumulation at the centrosome during the centrosome maturation stage of mitosis is known to depend on the adaptor protein NEDD1/GCP-WD and to be controlled by the kinase Plk1. Surprisingly, and although Plk1 binds and phosphorylates NEDD1 at multiple sites, the mechanism by which this kinase promotes centrosomal recruitment of γ-tubulin has remained elusive. Using Xenopus egg extracts and mammalian cells, the scientists found that Nek9, a kinase required for normal mitotic progression and spindle organization, phosphorylates NEDD1, driving its recruitment and thereby that of γ-tubulin to the centrosome. This role of Nek9 requires its activation by Plk1-dependent phosphorylation.

       Errors in chromosome distribution cause many spontaneous miscarriages, some genetic defects such as trisomies, and are related to the development of tumors. Nek9 exerts its action between two molecules, Plk1 and Eg5, of interest as antitumoral agents and for which inhibitors are already in advanced stages of clinical trials. Nek9 could well be added to the list of cell division target candidates.

      Without Nek9 the spindle would not form properly and cell division would be hindered, the cells would die or cause aneuploidies, with unequal distribution of chromosomes, an event that is common in tumors. 

(Source: Joan Roig et al., 2012. Nek9 Phosphorylation of NEDD1/GCP-WD Contributes to Plk1 Control of γ-Tubulin Recruitment to the Mitotic Centrosome. Current Biology)

The architecture of RNA polymerase fidelity

The basis for transcriptional fidelity by RNA polymerase is not understood, but the ‘trigger loop’, a conserved structural element that is rearranged in the presence of correct substrate nucleotides, is thought to be critical. A recent study sheds new light on the ways in which the trigger loop may promote selection of correct nucleotide triphosphate substrates

           Replicative or transcribing nucleic acid polymerases must produce complementary copies of nucleic acid templates by a mechanism that strikes a fine balance between fidelity and speed. For many of these enzymes, this is achieved by high selectivity for the correct substrate, with a proofreading step for removing incorrect nucleotides if the selection step fails.

           In broad terms, it is thought that binding of the correct complementary nucleotide to the DNA template in the RNAP active site induces closure of the site, with the correct alignment of critical amino acids for the polymerization reaction and thus efficient catalysis. The critical component in this structural rearrangement is the trigger loop, a flexible element of the largest subunit of RNA polymerase (the β’ subunit in eubacterial RNAP, and the Rpb1 subunit in eukaryotic RNA polymerase II (Pol II)) that interacts with the substrate and other elements of the enzyme active site. Removal of the trigger loop causes a drastic reduction in both the speed and the accuracy of nucleotide addition which is consistent with the general picture sketched above; and substitution mutants within the trigger loop can either increase or decrease the RNAP elongation rate in vitro in Escherichia coli or Pol II, suggesting selection for an optimum balance of speed with accuracy. The exact role of the trigger loop in selective binding and catalysis has, however, remained unclear.

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DNA has no reason to feel special : the discovery of XNA’s

Scientists have found that six polymer alternatives to DNA can pass on genetic information, and have evolved one type to specifically bind target molecules. They say that their work reveals both broader chemical possibilities for these key life functions and provides a powerful tool for nanotechnology and medicine. An international team of researchers has shown that artificial nucleic acids - called “XNAs” - can replicate and evolve, just like DNA and RNA.

          The researchers, led by Philipp Holliger and Vitor Pinheiro, synthetic biologists at the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK, say their findings have major implications in everything from biotherapeutics, to exobiology, to research into the origins of genetic information itself. This represents a huge breakthrough in the field of synthetic biology.

         A key hurdle for the team was to create enzymes that could copy a gene from a DNA molecule to an XNA molecule, and other enzymes that could copy it back into DNA. They started with enzymes that do this in DNA only. Over the years the team made incremental tweaks until they produced enzymes that could work on XNAs.

        ”There is no overwhelming functional imperative for life to be based on DNA or RNA,” says Phil Holliger from the MRC Laboratory of Molecular Biology in Cambridge, UK, who led the team. ‘Other polymers can perform these functions, at least at a basic level.’ Holliger’s team’s xeno-nucleic acid (XNA) polymers each replace DNA’s ribofuranose sugar ring with six other cyclic structures that can still form helical chains and base pairings. But rather than using relatively inefficient chemical synthesis, the scientists wanted to exploit polymerase and reverse transcriptase enzymes to copy genetic information from DNA templates to XNAs. In living organisms, polymerases can make RNA from nucleotide monomers using existing DNA strands as templates. Reverse transcriptases can then create a copy of the original DNA strand from that RNA in the same way.

“The immediate question is whether these XNAs can be introduced into cells,” says Farren Isaacs of Yale University in New Haven, Connecticut. Once the XNAs were installed, they could replicate and evolve on their own. “That would be remarkable.”

Source: Royal Society of Chemistry. 2012.

Further reading:

• Joyce, G. F. Toward an Alternative Biology. Science 336, 307-308 (2012).
• Pinheiro et al, 2012. Synthetic Genetic Polymers Capable of Heredity and Evolution. Science. 336, 341 (DOI: 10.1126/science.1217622)

Mutation Rates in Cancer Cells Strongly Linked to How Chromatin Is Organized

Inside our cells, DNA is packed in a dense structure called chromatin so the cell can replicate, repair any DNA damage during cell division, and control which genes are expressed. Researchers from the Center for Genomic Regulation CRG have found that chromatin has a lot to do with where mutations occur in the genome in cancer cells.

         Cancer is considered to be a genetic disease, with its leading cause the various mutations occurring while the genome is duplicated during cell division. Many genetic and epigenetic features have been proposed to influence the rate at which mutations occur along the genome. Researchers from the CRG have found that chromatin organization is the feature most strongly linked with mutation rates, at least in cancer cells.

          The researchers studied samples from different types of tissues and with different types of mutations in cancer cells like leukemia, melanoma, small lung cancer and prostate cancer. They obtained the data through open access repositories of genome databases. Since the first genome was sequenced, all genomic data from public funded research are supposed to freely available through these repositories. Among many other interests, one strong point of using data already collected by multiple other scientists is that biases are cancelled out because of the amount of experiments.

          The principal investigator, ICREA research professor Ben Lehner, says ‘Large-scale experiments such as the cancer genome projects mean that in biology it is now often possible to test an idea using data that has already been generated. The data from these projects can be used by groups worldwide to help us learn about the causes of cancer, but they can also be used to understand some basic problems in genetics such as why some regions of the genome mutate faster than others.

Source: Centre for Genomic Regulation (2012, July 26). 

Not just another genome

” Sequence analysis of the Daphnia pulex genome holds some surprises that could not have been anticipated from what was learned so far from other arthropod genomes. It establishes Daphnia as an eco-genetical model organism par excellence.”

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Genome of an aquatic sensor

               In a typical lake, Daphnia lives in paradise with food in the form of algae swimming around it ready to be collected at will. But no paradise lasts for ever. As the Daphnia population grows, algae become increasingly rare to the point of almost complete disappearance, leading to a short phase in the yearly cycle of a lake in which the water becomes crystal clear. Daphnia itself increasingly becomes a victim of predators during the yearly cycle and, although it starts to develop defenses, its population shrinks such that the algae can become more abundant again. The nutrient flux that is involved in this cycle is enormous and drives the whole ecology of the lake. But how can a genome sequence help to learn more about this ecology? The Daphnia genome turns out to hold a large number of genes which were previously not known and which, excitingly, are likely to have a specific role in the interaction with its environment. The Daphnia genome may thus become a Rosetta stone for studying the genetic repertoire of fresh water ecology

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Life cycle plasticity may facilitate rapid evolution

             Why is Daphnia so particularly amenable to developing an eco-responsive gene repertoire? On the one hand it is known that positive selection is much more efficient in large populations, since even genes or alleles that provide only a very small advantage are retained in the population rather than being lost by drift. However, large population sizes are also found in the insects for which full genome sequences are available - and they show no indication of a particularly strong genomic response to the environment. The explanation may lie with some additional peculiarities of population genetics shown by Daphnia.                        

              It can switch between parthenogenetic and sexual reproduction and it can produce resting stages that can survive for decades. Since parthenogenetic reproduction is numerically twice as efficient as sexual reproduction, Daphnia takes advantage of this in spring when a particularly fast population expansion is possible. This leads to a rapid amplification of clones that may have only a minimal advantage over their conspecifics. Of course, once the environment changes, the advantage of such clones may falter quickly, but this is the point where they can go into a sexual cycle and can produce special eggs that are protected by a cuticular structure that allows them to survive in the mud. Thus, all lakes harbor a genetic reservoir of resting eggs derived from animals that had a particular advantage at a previous time. Genes or alleles that were once successful can thus be preserved, even if the environmental conditions are temporarily changed. An explicit evolutionary theory that models the long-term adaptive consequences of such complex life cycles is still missing but, at least intuitively, it would seem that this adds to the evolutionary dynamics that have led to the special gene repertoire of Daphnia.

              Because of these peculiarities, Daphnia should now also become a prime model for studying the evolution and the role of sex. One of the companion papers has indeed already specifically addressed such issues by looking at the evolutionary dynamics of transposons in Daphnia [8]. These authors identified the major transposon families in the Daphnia genome and found active copies for most of them. Six of these were then studied in lines where sex was either promoted or inhibited. The data indicate that sexual reproduction is indeed a major factor to keep the elements under control. This effect could at least partially compensate for the short-term cost of sex and thus explain why sexual reproduction is maintained. Intriguingly, a previous study had suggested that sexual and parthenogenetic reproduction makes use of the same set of meiosis related genes and that an expansion of this gene complement may have helped to develop the parthenogenetic life cycle.

            Thus, both the ecological relevance and the evolutionary dynamics of Daphnia populations are bound to attract general attention to Daphnia as a new model system in genetics. The current genome paper focuses on D. pulex but another species of the genus, Daphnia magna, has an equally long history in ecological research and efforts to elucidate its genome are underway as well. These developments are bound to fuel the newly emerging discipline of ecological genomics, which has so far been one of the last black boxes of genetic research.