PLoS Genetics: New Articles

Genomic Hotspots for Adaptation: The Population Genetics of Müllerian Mimicry in the Heliconius melpomene Clade

2010-02-05T08:00:00Z

Author Summary

The diversity of wing patterns in Heliconius butterflies is a longstanding example of both Müllerian mimicry and adaptive radiation. The genetic regions controlling such patterns are “hotspots” for adaptive evolution, with small regions of the genome controlling major changes in wing pattern. Across multiple hybrid zones in Heliconius melpomene and related species, we no find no strong population signal of recent selection. Nonetheless, we find significant associations between genetic variation and wing pattern at multiple sites. This suggests patterning alleles are relatively old, and might be a better model for most natural adaptation, in contrast to the simple genetic basis of recent human-induced selection such as pesticide resistance. Strikingly, across the region controlling the red forewing band, a very strong association with phenotype implicates three genes as potentially being involved in control of wing pattern. One of these, a kinesin gene, shows parallel differences in expression levels between divergent forms in the two mimetic species, making it a strong candidate for control of wing pattern. These results show that mimicry involves parallel changes in gene expression and strongly suggest a role for this gene in control of wing pattern.

Genomic Hotspots for Adaptation: The Population Genetics of Müllerian Mimicry in Heliconius erato

2010-02-05T08:00:00Z

Author Summary

Identifying the genetic changes responsible for beneficial variation is essential for understanding how organisms adapt. Here, we use a combination of mapping, population genetic analysis, and gene expression studies to identify the genomic regions responsible for phenotypic evolution in the Neotropical butterfly Heliconius erato. H. erato, together with its co-mimic H. melpomene, have undergone parallel and concordant radiations in their warningly colored wing patterns across Central and South America. The “genes” underlying the H. erato color pattern radiation are classic examples of Mendelian loci of large effect and are under strong natural selection. Nonetheless, we do not see a clear molecular signal of recent natural selection, suggesting that the H. erato color pattern radiation, or the alleles that underlie it, may be quite old. Moreover, rather than being single locus, the genetic patterns suggest that multiple, widely dispersed loci may underlie pattern variation in H. erato. One of these loci, a kinesin gene, shows parallel expression differences between races during wing pattern formation in both H. erato and H. melpomene, suggesting that it plays an important role in pattern variation. High rates of recombination within naturally occurring H. erato hybrid zones mean that finer genetic dissection will allow us to localize causative sites and better understand the history and molecular basis of this extraordinary adaptive radiation.

Wing Patterns in the Mist

2010-02-05T08:00:00Z

Mutations in SLC29A3, Encoding an Equilibrative Nucleoside Transporter ENT3, Cause a Familial Histiocytosis Syndrome (Faisalabad Histiocytosis) and Familial Rosai-Dorfman Disease

2010-02-05T08:00:00Z

Author Summary

The histiocytoses are a group of systemic disorders usually confined to childhood and are caused by an excessive number of histiocytes which phagocytose other cells and process antigens. Although nearly a century has passed since histiocytic disorders were recognised, their pathophysiology remains largely unclear, and treatment is nonspecific. The identification of SLC29A3 mutations as the molecular basis for a familial form of syndromic histiocytosis (FHC/RDD) confirms a direct link between Faisalabad histiocytosis and Rosai-Dorfman disease and links these disorders to other SLC29A3-associated phenotypes.

DNA Binding of Centromere Protein C (CENPC) Is Stabilized by Single-Stranded RNA

2010-02-05T08:00:00Z

Author Summary

Here we address the issue of how genetic information is passed from one generation to the next without the involvement of specific DNA sequences. This type of inheritance is referred to as epigenetics. Centromeric sequences are highly variable and in many cases are not sufficient for centromere function. Rather, secondary features of the DNA, such as methylation or associated RNA molecules may serve to recruit key centromere binding proteins. Prior data from several species have established that single-stranded RNAs are surprisingly abundant on centromeric chromatin. Here we identified the DNA-binding domain of a key centromere binding protein in maize (CENPC) and showed that it requires single-stranded RNA to effectively bind DNA in vitro. When the DNA/RNA binding domain was deleted, the accuracy of CENPC targeting to centromeres was reduced but not abolished. The results bolster the view that centromere-bound RNA is one component of the epigenetic determination process that assures centromeres are stably inherited. In addition, our data suggest a general mechanism for how RNA can influence the binding of chromatin proteins to DNA.

Genetic and Functional Dissection of HTRA1 and LOC387715 in Age-Related Macular Degeneration

2010-02-05T08:00:00Z

Author Summary

Age-related macular degeneration (AMD) is the leading blindness cause in western countries. Several genes encoding components of the complement pathway—including CFH, C2/BF, and C3—have been confirmed to be associated with AMD, as well as a region on 10q26 that encompasses two genes. Recent data have suggested that loss of LOC387715 on 10q26, mediated by an insertion/deletion (in/del) at its 3'UTR that destabilizes its message, is causally related with the disorder. We found that a common disease haplotype including the in/del and rs11200638 also has an effect on the transcriptional upregulation of the adjacent gene, HTRA1. We propose a binary model where downregulation of LOC387715 and concomitant upregulation of HTRA1 best explain the risk associated with the 10q26 AMD region.

Replication and Active Demethylation Represent Partially Overlapping Mechanisms for Erasure of H3K4me3 in Budding Yeast

2010-02-05T08:00:00Z

Author Summary

Organisms can inherit information beyond DNA sequence, a phenomenon known as epigenetic inheritance. It is widely believed that chromatin marks provide a carrier for epigenetic information, a hypothesis that is less-supported than generally believed. In this study, we measure the erasure of a “memory” mark of active transcription, H3K4me3. We find that this signal-responsive chromatin mark largely returns to baseline levels within one generation. Furthermore, we find that this erasure occurs during S phase in a manner consistent with its loss during replication, yet we find that replication only contributes modestly to the erasure process. Instead, active enzymatic demethylation is required for erasure. Together, these results show that even chromatin states widely associated with epigenetic memory are only maintained in the ongoing presence of activating signals, and are not generally heritable.

Deletion of the Huntingtin Polyglutamine Stretch Enhances Neuronal Autophagy and Longevity in Mice

2010-02-05T08:00:00Z

Author Summary

Expansion of a stretch of glutamines near the amino-terminus of huntingtin (htt), the protein product of the IT15 gene, is a deleterious mutation that causes Huntington's disease (HD). Here we show, in contrast, that deletion of htt's normal polyglutamine stretch (ΔQ-htt) is a potentially beneficial mutation that can ameliorate HD mouse model phenotypes when ΔQ-htt is expressed together with a version of htt with the HD mutation. In addition, ΔQ-htt expression can enhance longevity when expressed in either an HD mouse model or in non–HD mice. ΔQ-htt's effects on both lifespan and HD model phenotypes are likely due to an increase in autophagy, a major recycling pathway in cells that is involved in the turnover of cellular components, and aggregated protein. Based on our results, we suggest that development of therapeutic agents that can stimulate autophagy may help both in treating neurodegenerative disorders like HD and also in increasing longevity.

A Kinase-Independent Role for the Rad3ATR-Rad26ATRIP Complex in Recruitment of Tel1ATM to Telomeres in Fission Yeast

2010-02-05T08:00:00Z

Author Summary

ATM and ATR kinases are two evolutionarily conserved sensors of DNA damage, responsible for maintaining stable genomes in all eukaryotic cells. These two kinases safeguard eukaryotic genomes against undesired double-stranded DNA breaks (DSBs) and errors during duplication of genomic DNA. Furthermore, ATM and ATR are redundantly required for stable maintenance of telomeres, protective structures at ends of linear eukaryotic chromosomes. Our current study in fission yeast demonstrates that the previously defined C-terminal Tel1^ATM interaction domain of the DNA repair protein Nbs1, which contributes to recruitment of Tel1^ATM to DSBs, is dispensable for recruitment of Tel1^ATM to telomeres, due to a previously unrecognized kinase-independent role of ATR in recruitment of Tel1^ATM to telomeres. Furthermore, the N-terminus of Nbs1 was found to be critical for recruitment of both ATR and ATM to telomeres. Regulators of telomere maintenance have recently emerged as potentially important therapeutic targets against tumorigenesis and aging in mammalian cells. Since proteins responsible for proper maintenance of telomeres and cellular responses to DNA damage are highly conserved between fission yeast and mammalian cells, a newly uncovered molecular crosstalk between ATM and ATR might also play critical roles in telomere maintenance and DNA damage responses in mammalian cells.

Human and Non-Human Primate Genomes Share Hotspots of Positive Selection

2010-02-05T08:00:00Z

Author Summary

An advantageous mutation spreads from generation to generation in a population until individuals that carry it, because of their higher reproductive success, completely replace those that do not. This process, commonly known as positive Darwinian selection, requires the selected mutation to induce a new non-neutral heritable phenotypic trait, and this has been shown to occur unexpectedly frequently during recent human evolution. Although the exact advantageous mutation is difficult to identify, it leaves a wider footprint on neighbouring linked neutral variation called a selective sweep. We have developed an empirical method that uses whole-genome shotgun sequences of single individuals to detect selective sweeps. By doing so, we were able to extend to chimpanzee, orangutan, and macaque individuals analyses of recent positive selection that until now were only available for human. Comparisons of genes candidates for positive selection between human and non-human primates then revealed an unexpectedly high number of cases where a selective sweep at a gene in humans is mirrored by independent positive selection at the same gene in multiple other primates. This result has future implications for understanding the nature of biological changes that underlie selective sweeps in humans.

PLoS Genetics Issue Image | Vol. 6(1) January 2010

2010-01-29T08:00:00Z

A panicle of the rice pseudoviviparous mutant shows that all spikelets are replaced by vigorous juvenile plantlets.

In this issue of PLoS Genetics, Wang et al. report a spontaneous pseudoviviparous mutant phoenix (pho) in rice. In pho plants, all spikelets are replaced by young plantlets, and the reproductive strategy is completely changed from sexual to asexual. Further analyses revealed that pho is caused by mutations of two MADS-box transcription factors. These findings provide an insight into the mechanism of pseudovivipary in plants.

Image Credit: Zhukuan Cheng (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China)

Genetic Crossovers Are Predicted Accurately by the Computed Human Recombination Map

2010-01-29T08:00:00Z

Author Summary

In eukaryotes genetic crossovers are responsible for generating genetic diversity and ensuring the proper segregation of chromosomes. Genetic crossovers are tightly clustered in hotspots. Although the existence of hotspots in humans is clearly proven, mechanisms of their formation and the regulation of meiotic recombination in general remain poorly understood. An additional complication in studies of meiotic recombination is the fact that the direct experimental mapping of human hotspots on a genome-wide scale is not feasible with current methods. The best available indirect methods compute the position of hotspots from patterns of historic associations between genetic markers in population samples. In this study we determined the positions of genetic crossovers in ten pedigrees of European origin and then compared the positions of crossovers with the hotspots computed from HapMap data. Importantly, we find that the population-averaged computed map is in close agreement with the observed distribution of genetic crossovers. We also find that cryptic hotspots that are not easily detected in the computed European map can be more effectively identified if other populations are included in the analysis. Our analysis shows that high-resolution recombination profiles are highly similar between distantly related populations and that by including computed hotspots from several populations we can predict nearly all crossovers.

U87MG Decoded: The Genomic Sequence of a Cytogenetically Aberrant Human Cancer Cell Line

2010-01-29T08:00:00Z

Author Summary

Glioblastoma has a particularly dismal prognosis with median survival time of less than fifteen months. Here, we describe the broad genome sequencing of U87MG, a commonly used and thus well-studied glioblastoma cell line. One of the major features of the U87MG genome is the large number of chromosomal abnormalities, which can be typical of cancer cell lines and primary cancers. The systematic, thorough, and accurate mutational analysis of the U87MG genome comprehensively identifies different classes of genetic mutations including single-nucleotide variations (SNVs), insertions/deletions (indels), and translocations. We found 2,384,470 SNVs, 191,743 small indels, and 1,314 large structural variations. Known gene models were used to predict the effect of these mutations on protein-coding sequence. Mutational analysis revealed 512 genes homozygously mutated, including 154 by SNVs, 178 by small indels, 145 by large microdeletions, and up to 35 by interchromosomal translocations. The major mutational mechanisms in this brain cancer cell line are small indels and large structural variations. The genomic landscape of U87MG is revealed to be much more complex than previously thought based on lower resolution techniques. This mutational analysis serves as a resource for past and future studies on U87MG, informing them with a thorough description of its mutational state.

Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

2010-01-22T08:00:00Z

Author Summary

Double strand DNA breaks (DSBs) are dangerous chromosomal lesions that can lead to genome rearrangements, genetic instability, and cancer if not accurately repaired. Eukaryotes activate a surveillance mechanism, called DNA damage checkpoint, to arrest cell cycle progression and facilitate DNA repair. Several factors are physically recruited to DSBs, and specific kinases phosphorylate multiple targets leading to checkpoint activation. Budding yeast is a good model system to study checkpoint, and most of the factors involved in the DSBs response were originally characterized in this organism. Using the yeast Saccharomyces cerevisiae, we explored the functional role of polo kinase Cdc5 in regulating the DSB–induced checkpoint. Polo kinases have been previously involved in checkpoint inactivation in all the eukaryotes, and they are frequently overexpressed in cancer cells. We found that elevated levels of Cdc5 affect the cellular response to a DSB at different steps, altering DNA processing and overriding the signal triggered by checkpoint kinases. Our findings suggest that Cdc5 likely regulates multiple factors in response to a DSB and provide a rationale for a proteome-wide screening to identify targets of polo kinases in yeast and human cells. Such information may have a practical application to design specific molecular tools for cancer therapy. Two related papers published in PLoS Biology—by Vidanes et al., doi:10.1371/journal.pbio.1000286, and van Vugt et al., doi:10.1371/journal.pbio.1000287—similarly investigate the phenomenon of checkpoint adaptation/overriding.

The Gift of Observation: An Interview with Mary Lyon

2010-01-22T08:00:00Z

DEP and AFO Regulate Reproductive Habit in Rice

2010-01-22T08:00:00Z

Author Summary

Sexual reproduction is essential for the life cycle of most flowering plants. However, pseudovivipary, in which floral organs are replaced by bulbils or plantlets, provides an asexual means for many grasses to reproduce in extreme environments. Although the molecular mechanism of pseudovivipary is still unknown, the high-frequency occurrence of pseudovivipary in extreme environments indicates that only a few key regulators are responsible for the switch of reproductive habit. Here, by analyzing three naturally occurring mutants in rice, we show that mutations in DEP and AFO lead to the transformation of rice flowers/spikelets into juvenile plantlets and subsequently the switch of reproductive strategy from sexual to asexual, suggesting that DEP and AFO might work cooperatively to regulate reproductive habit in rice. Thus, we reveal a critical mechanism of the switch of reproductive habit in plants. In addition, our results also make it possible to manipulate the reproductive habit of plants, at least in rice.

Nonsense-Mediated Decay Enables Intron Gain in Drosophila

2010-01-22T08:00:00Z

Author Summary

The surprising observation 30 years ago that genes are interrupted by non-coding introns changed our view of gene architecture. Intron number varies dramatically among species; ranging from nine introns/gene in humans to less than one in some simple eukyarotes. Here we ask where new introns come from and how they are maintained in a population. We find that novel introns do not arise from pre-existing introns, although the mechanisms that generate novel introns remain unclear. We also show that novel introns carry only weak signals for their identification and removal, and therefore depend on nonsense-mediated decay (NMD). NMD maintains RNA quality control by degrading transcripts that have not been spliced properly. We propose that NMD shelters novel introns from natural selection. This increases the likelihood that a novel intron will rise in frequency and be maintained within a population, thus increasing the rate of intron gain.

The Caenorhabditis elegans Elongator Complex Regulates Neuronal α-tubulin Acetylation

2010-01-22T08:00:00Z

Author Summary

We were able to demonstrate how a screen, that utilized the nematode model organism Caenorhabditis elegans, yielded the novel discovery that the Elongator protein complex is critical for neuronal development and microtubule acetylation. Other scientists have previously shown that the Elongator is important for transcription, but also hypothesized that a hitherto unknown function within the cytoplasm prevails. Regulation of microtubule acetylation is indeed important for cellular function. Our results provide the first tantalizing insights of how worms display neuronal phenotypes that may be linked to changes in degrees of acetylation of microtubules. We also point out that the Elongator itself has a defined acetyltransferase function that could well be directly responsible for the acetylation of α-tubulin. In conclusion, we discuss how the regulation of microtubule acetylation might impact on the understanding of human neurodegenerative diseases, namely Familial Dysautonomia and Amyotrophic Lateral Sclerosis.

Environmental and Genetic Determinants of Colony Morphology in Yeast

2010-01-22T08:00:00Z

Author Summary

Baker's yeast forms smooth round colonies when grown in favorable conditions. When starved for one or more nutrients, yeast can alter its growth pattern to produce complex structures consisting of numerous interacting cells. One mode of growth, the colony morphology response, produces visually striking, lacy colony architectures. We describe both conditions that induce this morphology and also genes and pathways that are required for the response. We demonstrate that low levels of carbon combined with abundant nitrogen trigger complex colony formation. Using a candidate gene approach coupled with genome-wide mutagenesis, we identified genes involved in the production of complex colony morphology. Many of these genes are components of either a MAP kinase cascade or the Ras-cAMP-PKA pathway, two well-studied signaling pathways that are conserved across eukaryotic organisms. Yeast use these pathways to mediate cellular responses to changes in their environment. We observe shared characteristics between complex colonies and biofilms, which are organized communities of microorganisms with relevance to human health and human infrastructure, making colony morphology a candidate model for understanding how microorganisms interact to form complex structures.

Distinct Type of Transmission Barrier Revealed by Study of Multiple Prion Determinants of Rnq1

2010-01-22T08:00:00Z

Author Summary

Prions, self-propagating protein conformations and causative agents of lethal neurodegenerative diseases, present a serious public health threat: they can arise sporadically and then spread by transmission to the same, as well as other, species. The risk of infecting humans with prions originating in wild and domestic animals is determined by the so-called transmission barriers. These barriers are attributed to differences in prion proteins from different species, but their underlying mechanisms are not clear. Recent findings that the prion state is transmitted through the interaction between short transmitting regions within prion domains revealed one type of transmission barrier, where productive templating is impeded by non-matching amino acids within transmitting regions. Here we present studies of the prion domain of the [PIN⁺]-forming protein, Rnq1, and describe a distinct type of transmission barrier not involving individual amino acid mismatches in the transmitting regions. Rnq1's prion domain is complex and encompasses four regions that can independently transmit the prion state. Our data suggest that multiple prion determinants of a complex prion domain act cooperatively to attain the prion conformation, and transmission barriers occur between protein variants that cannot form the same higher order structure, despite the identity of the region(s) driving the transmission.

Evidence for Pervasive Adaptive Protein Evolution in Wild Mice

2010-01-22T08:00:00Z

Author Summary

The prevalence of natural selection at the DNA level remains a controversial issue in evolutionary biology. In particular, estimates of the proportion of adaptive amino acid changes (α) vary greatly between taxa, being 50% or more in bacteria and fruit flies, but at most 13% in hominids. Here, we infer the frequencies of polymorphisms in protein-coding genes of 15 Mus musculus castaneus individuals sampled from the ancestral range of the house mouse species complex. By combining the polymorphism data with nucleotide divergence to the related murid species M. famulus and the rat, we obtain an estimate for α of 57%. This represents the first estimate of α for a mammal other than humans. The high rate of adaptive protein evolution in wild mice and other taxa implies that hominids may be somewhat unusual in having low rates of adaptive protein evolution. One possible cause of this is the low effective population size in humans, which is predicted to lead to less effective natural selection and fewer adaptive mutations. This is consistent with the higher frequency of nearly neutral deleterious amino acid mutations in hominids than murids that we infer in our analysis.

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

2010-01-22T08:00:00Z

Author Summary

The repair of DNA breaks in cells is critical for maintaining genomic integrity and suppressing tumor development. DNA breaks can arise from exogenous agents such as ionizing radiation (IR) or can form during the process of germ cell (sperm and egg) generation. BRIT1 protein (also known as MCPH1) is a recently identified DNA damage responding protein, and its mutations or reduced expression are found in primary microcephaly (small brain) patients, as well as in cancer patients. To investigate BRIT1's physiological functions and dissect the underlying molecular mechanism, we used a genetic approach (gene targeting technology) to delete BRIT1 gene in mice and generated a mouse model with BRIT1 deficiency (called BRIT1-knockout mice). Here, we showed that BRIT1 knockout mice are more sensitive to IR due to their inability to repair the IR-induced DNA breaks. These mice are also infertile, and their DNA repair during the process of germ cell generation was impaired substantially. Thus, in this study, we generated a novel mouse model (BRIT1 knockout mice) with striking phenotypes related to defective DNA repair and clearly demonstrated the essential role of BRIT1 in DNA repair at organism level.

Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair

2010-01-22T08:00:00Z

Author Summary

Induction of double-strand breaks (DSBs) in chromosomal DNA effectively activates a program of cellular suicide and is widely used for chemotherapy on malignant cancer cells. Cells resist such therapies by quickly repairing the DSBs. Repair is carried out by two major DSB repair pathways, homologous recombination (HR) and nonhomologous end-joining. However, these pathways cannot join DSBs if their ends are chemically modified, as seen in the DSB ends that would arise after the prolonged treatment of the cells with topoisomerase inhibitors such as camptothecin and etoposide. These anti-cancer drugs can produce the polypeptides covalently attached to the 3′ or 5′ end of DSBs. It remains elusive which enzymes eliminate these chemical modifications prior to repair. We here show evidence that the BRCA1-CtIP complex plays a role in eliminating this chemical modification, thereby facilitating subsequent DSB repair. Thus, BRCA1 and CtIP have dual functions: their previously documented roles in HR and this newly identified function. This study contributes to our ability to predict the effectiveness of chemotherapeutic agents prior to their selection by evaluating the activity of individual repair factors. Accurate prediction is crucial, because chemotherapeutic agents that cause DNA damage have such strong side effects.

Evolutionary Mirages: Selection on Binding Site Composition Creates the Illusion of Conserved Grammars in Drosophila Enhancers

2010-01-22T08:00:00Z

Author Summary

Because mutation is a random process, most biologists assume that apparently non-random features of genome sequences must be the result of natural selection acting to create and preserve them. Where this is true, genome sequences provide a powerful means to infer aspects of molecular, cellular, and organismal biology from the signatures of selection they have left behind. However, recent analyses have shown that many aspects of genome structure and organization that have traditionally been attributed to selection can often arise from random processes. Several groups—including ours—studying the sequences that specify when and where genes should be produced have identified common, seemingly conserved, architectural features, based on which we have proposed new models for the activity of the complex molecular machines that regulate gene expression. However, in the work described here we simulate the evolution of these regulatory sequences and show that many of the features that we and others have identified can arise as a byproduct of random mutational processes and selection for other properties. This calls into question many conclusions of comparative genome analysis, and more generally highlights what Michael Lynch has called the “frailty of adaptive hypotheses” for the origins of complex genomic structures.

Differential Localization and Independent Acquisition of the H3K9me2 and H3K9me3 Chromatin Modifications in the Caenorhabditis elegans Adult Germ Line

2010-01-22T08:00:00Z

Author Summary

Histone methylation is a widespread feature of eukaryotic chromatin and has been implicated in numerous aspects of chromosome function. Methyl marks have been noted to occur on numerous different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. In this work we investigate the relationships between the dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3). Our analysis exploits organizational features of the C. elegans germ line that facilitate cytological visualization of distinct chromosomal features and dynamic changes in localization that are associated with different chromatin marks. Despite the fact that H3K9me2 and H3K9me3 modify the same amino acid residue, our work reveals that these marks exhibit very different localization patterns in the adult C. elegans germ line and become concentrated on chromatin with different properties. Moreover, we show that these marks are acquired independently, requiring different histone methyltransferases, implying that H3K9me3 is not built upon the H3K9me2 mark. Collectively, our data indicate that H3K9me2 and H3K9me3 are highly autonomous chromatin modifications, functioning independently of one another in the C. elegans germ line.

A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

2010-01-15T08:00:00Z

Author Summary

Deinococcus radiodurans bacterium is among the best-known organisms found to resist extremely high exposures to desiccation and ionizing radiation, both causing extensive DNA double-strand breaks. Because a single unrepaired DNA double-strand break is usually lethal, DNA double-strand breaks are considered as the most severe form of genomic damage. The extreme radioresistance of D. radiodurans is linked to its ability to reconstruct a functional genome from hundreds of chromosomal fragments. Genome reconstitution occurs through a two step process: (i) an extended synthesis-dependent strand-annealing process (ESDSA) that assembles genomic fragments in long linear intermediates that are then (ii) processed through recombination to generate circular chromosomes. Here, we demonstrate the essential role of key components of the D. radiodurans RecF pathway in ESDSA. We show that (i) inactivation of only one exonuclease (RecJ) results in cell lethality; (ii) cells devoid of RecF, RecO, or RecR display greatly impaired growth; (iii) RecF, RecO, or RecR proteins are essential for radioresistance through ESDSA; and (iv) UvrD helicase has an unexpected crucial function in DNA double-strand-break repair through ESDSA.

Irradiation-Induced Deinococcus radiodurans Genome Fragmentation Triggers Transposition of a Single Resident Insertion Sequence

2010-01-15T08:00:00Z

Author Summary

Induction of transposition in prokaryotes under cell stress conditions is potentially important in creating diversity facilitating adaptation to severe environments. In Deinococcus radiodurans, the most radiation-resistant organism known, despite abundance of resident insertion sequences (IS), transposition of a single IS, ISDra2, was found to be strongly induced by irradiation. We show that both steps involved in transposition, IS excision, and insertion, increase significantly following host cell irradiation and, using PCR analysis of genomic DNA, that exposure to γ-irradiation stimulates massive excision of the single genomic ISDra2 copy as a DNA circle and reclosure of the empty site. These events are closely correlated with the initiation of the process leading to genome reassembly from chromosomal fragments, which occurs mainly through a mechanism generating long stretches of single-stranded DNA. Consistent with this, we also demonstrate a requirement for single strand DNA substrates in TnpA-catalysed cleavage and strand transfer in vitro. Since we find no evidence for irradiation-induced expression of the ISDra2 transposase, we infer that transposition is triggered by the increase in its single-strand DNA substrate. The potential impact on genome reassembly and in creating genome host diversity by triggering transposition in this way is discussed.

The Systemic Imprint of Growth and Its Uses in Ecological (Meta)Genomics

2010-01-15T08:00:00Z

Author Summary

Microbial minimal generation times vary from a few minutes to several weeks. The reasons for this disparity have been thought to lie on different life-history strategies: fast-growing microbes grow extremely fast in rich media, but are less capable of dealing with stress and/or poor nutrient conditions. Prokaryotes have evolved a set of genomic traits to grow fast, including biased codon usage and transient or permanent gene multiplication for dosage effects. Here, we studied the relative role of these traits and show they can be used to predict minimal generation times from the genomic data of the vast majority of microbes that cannot be cultivated. We show that this inference can also be made with incomplete genomes and thus be applied to metagenomic data to test hypotheses about the biomass productivity of biotopes and the evolution of microbiota in the human gut after birth. Our results also allow a better understanding of the co-evolution between growth rates and genomic traits and how they can be manipulated in synthetic biology. Growth rates have been a key variable in microbial physiology studies in the last century, and we show how intimately they are linked with genome organization and prokaryotic ecology.

Kidney Development in the Absence of Gdnf and Spry1 Requires Fgf10

2010-01-15T08:00:00Z

Author Summary

Kidney development requires the secreted protein GDNF, which signals via its cellular receptor RET to promote growth and branching of the ureteric bud, the progenitor of the collecting duct system. The transcription factors ETV4 and ETV5 regulate gene expression in response to GDNF. We report that deleting Spry1, a feedback inhibitor downstream of RET, largely rescues kidney development in mice lacking GDNF or RET, although not in those lacking ETV4 and ETV5. Thus, GDNF and RET become dispensable in the absence of SPRY1, when their roles can be largely assumed by other signals and receptors, while ETV4 and ETV5 remain indispensible. We identify FGF10 as the signal responsible for kidney development in the combined absence of GDNF/RET signaling and SPRY1 negative regulation. But while the ureteric bud branches extensively in Gdnf−/−;Spry1−/− and Ret−/−;Spry1−/− kidneys, its pattern of branching is severely perturbed. This points to a unique function of GDNF in ureteric bud patterning.

Co-Orientation of Replication and Transcription Preserves Genome Integrity

2010-01-15T08:00:00Z

Author Summary

An important feature of genome organization is that transcription and replication are selectively co-oriented. This feature helps to avoid conflicts between head-on replication and transcription. The precise consequences of the conflict and how it affects genome organization remain to be understood. We previously found that reversing the transcription bias slows replication in the Bacillus subtilis genome. Here we engineered new inversions to avoid changes in other aspects of genome organization. We found that the reversed transcription bias is sufficient to decrease replication speed, and it results in lowered fitness of the inversion strains and a competitive disadvantage relative to wild-type cells in minimal medium. Further, by analyzing genomic copy-number snapshots to obtain replication speed as a function of genome position, we found that inversion of the strongly-transcribed rRNA genes obstructs replication during growth in rich medium. This confers a strong growth disadvantage to cells in rich medium, turns on DNA damage responses, and leads to cell death in a subpopulation of cells, while the surviving cells are more sensitive to genotoxic agents. Our results strongly support the hypothesis that evolution has favored co-orientation of transcription with replication, mainly to avoid these effects.

Maternal Ethanol Consumption Alters the Epigenotype and the Phenotype of Offspring in a Mouse Model

2010-01-15T08:00:00Z

Author Summary

In humans it has been known for some time that exposure to environmental insults during pregnancy can harm a developing fetus and have life-long effects on the individual's health. A well known example is fetal alcohol syndrome, where the children of mothers that consume large amounts of alcohol during pregnancy exhibit growth retardation, changes to the shape and size of the skull, and central nervous system defects. At present the molecular events underlying fetal alcohol syndrome are unknown. We have developed a model of alcohol exposure in the mouse, in which the genetics and the environment can be strictly controlled. We find that chronic exposure of the fetus to a physiologically relevant amount of alcohol during the first half of pregnancy results in epigenetic changes at a sensitive reporter gene and produces fetal alcohol syndrome-like features in some mice. Our model is a useful tool to study the underlying causes of fetal alcohol syndrome, and our work raises the interesting possibility that the long-term physical effects of alcohol exposure during pregnancy are mediated by epigenetic changes established in the fetus and then faithfully remembered for a lifetime. In the future, such epigenetic changes could be used as markers for the preclinical diagnosis and treatment of fetal alcohol spectrum disorders.

Altered Gene Expression and DNA Damage in Peripheral Blood Cells from Friedreich's Ataxia Patients: Cellular Model of Pathology

2010-01-15T08:00:00Z

Author Summary

Friedreich's ataxia is an inherited disease that causes progressive damage to the nervous system and affects the muscles and heart. The disease is caused by a defect in the frataxin gene, which is involved in iron homeostasis and likely protects against reactive oxygen species. In order to identify mechanisms involved in the nature and progression of the disease, we performed transcriptional profiling and measurements of mitochondrial and nuclear DNA damage on blood cells from FRDA patients. Transcriptional profiling was performed on blood samples from a cohort of 28 children compared to a control group. These data were then validated with a cohort of 14 adults with FRDA compared to a second independent control group. DNA damage was assessed on the blood samples from the 28 FRDA children, plus an additional 19 affected children, by quantitative PCR (QPCR). Transcriptional profiling revealed changes in gene expression consistent with the presence of genotoxic stress in FRDA patients. This finding was supported by the direct evidence that FRDA patients accumulated significantly higher levels of mitochondrial and nuclear DNA damage as compared to controls. The identification of potential biomarkers, including the DNA damage found in peripheral blood, may help identify therapeutic approaches for this devastating disease.

A Comprehensive Map of Insulator Elements for the Drosophila Genome

2010-01-15T08:00:00Z

Author Summary

The spatiotemporal specificity of gene expression is controlled by interactions among regulatory proteins, cis-regulatory elements, chromatin modifications, and genes. These interactions can occur over large distances, and the mechanisms by which they are controlled are poorly understood. Insulators are DNA sequences that can both block the interaction between regulatory elements and genes, as well as block the spread of regions of modified chromatin. To date, relatively few insulators have been identified in developing Drosophila embryos. We here present the genome wide identification of over 14,000 binding sites for 6 insulator-associated proteins. We demonstrate the existence of two broad classes of insulators. Insulators of both classes are enriched at the boundaries of a particular chromatin modification. However, only insulators bound by BEAF-32, CP190, and dCTCF are enriched in regions of open chromatin or demarcate gene boundaries, with a particular enrichment between differentially expressed promoters. Furthermore, insulators of this class are enriched at points of chromosomal rearrangement among the 12 species of sequenced Drosophila, suggesting that insulator defined regulatory boundaries are evolutionarily conserved.

Rising from the Ashes: DNA Repair in Deinococcus radiodurans

2010-01-15T08:00:00Z

Genetic Dissection of Differential Signaling Threshold Requirements for the Wnt/β-Catenin Pathway In Vivo

2010-01-15T08:00:00Z

Author Summary

Germline or somatic mutations in genes are the underlying cause of many human diseases, most notably cancer. Interestingly though, even in situations where every cell of every tissue of an organism carries the same mutation (as is the case for germline mutations), some tissues are more susceptible to the development of disease over time than others. For example, in familial adenomatous polyposis (FAP), affected persons carry different germline mutations in the APC gene and are prone to developing cancers of the colon and the rectum—and, less frequently, cancers in other tissues such as stomach, liver, and bones. Here we utilize a panel of mutant mice with truncating or hypomorphic mutations in the Apc gene, resulting in different levels of activation of the Wnt/β-catenin pathway. Our results reveal that different pathophysiological outcomes depend on different permissive signaling thresholds in embryonic, intestinal, and liver tissues. Importantly, we demonstrate that reducing Wnt pathway activation by 50% is enough to prevent the manifestation of embryonic abnormalities and disease in the adult mouse. This raises the possibility of developing therapeutic strategies that modulate the activation levels of this pathway rather than trying to “repair” the mutation in the gene itself.

The MCM-Binding Protein ETG1 Aids Sister Chromatid Cohesion Required for Postreplicative Homologous Recombination Repair

2010-01-15T08:00:00Z

Author Summary

DNA replication is a highly complex process and the source of potential DNA damage. It is of utmost importance that the damaged DNA is repaired before cells proceed through mitosis, because the genome holds all the information required for correct development. DNA replication results in two identical sister chromatids. A trick applied by cells to overcome damaged DNA is homologous recombination, using the undamaged copy of the sister chromatid as a template to repair the damaged one. This process is aided by keeping the two sister chromatids in close proximity after the replication process by the deposition of a molecular glue, called cohesin. In the present work, we identified the Arabidopsis thaliana ETG1 protein as a novel evolutionarily conserved replication factor that is needed for maintaining the sister chromatids physically aligned. In plants without ETG1, DNA damage builds up due to inefficient DNA repair. As a consequence, cell division is impaired with a huge impact on plant growth, highlighting the importance of cohesin for the correct development of eukaryotic organisms. Cohesion phenotypes observed upon the depletion of the orthologous human ETG1 protein indicate equally prominent roles for this particular factor during mammalian development.

Non-Coding Changes Cause Sex-Specific Wing Size Differences between Closely Related Species of Nasonia

2010-01-15T08:00:00Z

Author Summary

The regulation of cell size and cell numbers is an important part of determining the size of organs in development, as well as of controlling cell over-proliferation in diseases such as cancer and diabetes. How the regulation of cell size and number can change to produce different organ sizes is not well understood. Here, we investigate the recent evolution of sex-specific wing size differences between two species that involve changes to cell size and number regulation. Males of the emerging genetic model wasp Nasonia vitripennis have small wings and do not fly, while males of the closely related species N. giraulti have large wings and do fly. We isolated a locus that contributes substantially to this wing size difference by increasing cell size and cell number. Surprisingly, we found that the determinant for this wing size difference is located in the non-coding region between two known transcription factors, the master sex determining gene doublesex and neurogenesis regulator prospero. The mechanism by which ws1 regulates sex specific wing growth has yet to be determined, although differences in dsx expression level in developing male wings may indicate a role for this sex determination locus.