PLoS Genetics: New Articles

Regulation of Lifespan, Metabolism, and Stress Responses by the Drosophila SH2B Protein, Lnk

2010-03-19T07:00:00Z

Author Summary

Many human populations are experiencing increased life expectancy, and as populations age the incidence of age-related diseases becomes more prevalent. The identification of single gene mutations that extend lifespan in invertebrate model organisms has revealed that several cellular signaling pathways, including the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway, play a crucial role in modulating the ageing process across multiple species. Thus, studies carried out in yeast, worms, and flies have revealed evolutionarily conserved mechanisms of ageing, which are likely to be relevant to mammals, including humans. A recent study in Drosophila identified the SH2B family adaptor protein, Lnk, as an important regulator of the IIS pathway during organismal growth. In this study, we show that Lnk is also required to determine normal lifespan in Drosophila, as mutations that disrupt Lnk activity result in increased lifespan. In addition, these mutants show improved survival under conditions of stress and metabolic disregulation. Furthermore, we show that the expression of Lnk is regulated by the IIS responsive transcription factor, dFoxo. Our data therefore provide new mechanistic insights into the role of the IIS pathway in ageing.

HAP2(GCS1)-Dependent Gamete Fusion Requires a Positively Charged Carboxy-Terminal Domain

2010-03-19T07:00:00Z

Author Summary

Recent studies suggest that HAP2(GCS1) is a deeply conserved protein required for gamete membrane fusion, a critical yet poorly understood step in sexual reproduction. HAP2(GCS1) is present in many plant, protist, and animal genomes, and has been shown to be essential for fertilization in Arabidopsis, Chlamydomonas, and Plasmodium. The loss-of-function phenotype in Chlamydomonas suggests a direct role in gamete plasma membrane fusion. HAP2(GCS1) has no known functional domains, making it difficult to predict how it contributes to gamete fusion. We set out to map the critical features of this protein by testing a series of deletions, substitutions, and interspecific chimeras for their ability to rescue the hap2-1 fertilization defect in Arabidopsis. We found that the N-terminus does not tolerate sequence divergence, but the histidine-rich C-terminus does. We propose that the N-terminus of HAP2(GCS1) functions in part by interacting with proteins on the surface of female gametes. The key feature of the C-terminus is positive charge, a characteristic that could favor interactions with the plasma membrane that promote membrane fusion. Our studies provide a description of HAP2(GCS1) functional domains and provide an important framework for defining the role of this essential component of a conserved reproductive mechanism.

Multiple Signals Converge on a Differentiation MAPK Pathway

2010-03-19T07:00:00Z

Author Summary

Signal integration is an essential feature of information flow through signal transduction pathways. The mechanisms by which signals from multiple pathways become integrated into a coordinated response remain unclear. We show that multiple pathways that regulate filamentous growth converge on a differentiation-dependent MAPK pathway. Our findings indicate that more extensive communication occurs between signaling pathways that control the filamentation response than has previously been appreciated. We suggest that global communication hierarchies regulate information flow in other systems, particularly higher eukaryotes where multiple pathways typically function simultaneously to modulate a complex response.

Identification and Functional Analysis of the Vision-Specific BBS3 (ARL6) Long Isoform

2010-03-19T07:00:00Z

Author Summary

Retinitis pigmentosa (RP), a disorder of retinal degeneration resulting in blindness, occurs due to mutations in dozens of different genes encoding proteins with highly diverse functions. To date, there are no effective therapies to delay or arrest retinal degeneration. RP places a large burden on affected families and on society as a whole. We have studied a syndromic form of RP known as Bardet-Biedl Syndrome (BBS), which leads to degeneration of the photoreceptor cells and is associated with non-vision abnormalities including obesity, hypertension, diabetes, and congenital abnormalities of the kidney, heart, and limbs. In this study we utilized two model systems, the zebrafish and mouse, to evaluate the function of a specific form of BBS (BBS3). We have identified a novel protein product of the BBS3 gene and demonstrated that functional and structural abnormalities of the eye occur when this form of BBS3 is absent. This finding is of significance because it indicates that BBS3 mutations can lead to non-syndromic blindness, as well as blindness associated with other clinical features. This work also indicates that treatment of BBS3 blindness will require replacement of a specific form of the BBS3 gene.

Parental Genome Dosage Imbalance Deregulates Imprinting in Arabidopsis

2010-03-19T07:00:00Z

Author Summary

In mammals and plants, imprinted genes are expressed preferentially by the copy inherited from either the mother or the father. In plants genome dosage is easily manipulated using tetraploid plants that contain twice the genome dosage of the natural diploid plants. The increased maternal dosage reduces seed size while increased paternal dosage has the opposite effect. It was further proposed that parental genomic imbalances are directly mirrored by antagonistic regulations of imprinted genes encoding maternal growth inhibitors and paternal growth enhancers. However these hypotheses were never tested directly. We measured the expression of imprinted genes and their regulators, in crosses between diploid and tetraploid Arabidopsis plants. Surprisingly, parental dosage imbalance affected each imprinted gene in a different manner and the imprinted status was also affected. Our results point to a relationship between imprinting and dosage imbalance that is more complex than predicted.

Deciphering Normal Blood Gene Expression Variation—The NOWAC Postgenome Study

2010-03-12T08:00:00Z

Author Summary

As a major defence and transport system, blood cells are capable of adjusting gene expression in response to various clinical, biochemical, and pathological conditions. Here, we expand our understanding about the nature and extent of variation in gene expression from blood among healthy individuals. Using a large representative sample of postmenopausal women (N = 286) in the Norwegian Women and Cancer (NOWAC) postgenome study, we investigated blood gene expression changes due to normal inter-individuality (age, body mass index, fasting status), and exposure variables (smoking, hormone therapy, and medication use) at proportions and levels found in real life situations. Host genes were found to vary by inter-individual (i.e. fasting, BMI) and exposure (i.e. smoking) factors, and these gene lists may be used as a basis for further hypothesis development. Our study also establishes the feasibility of blood gene expression profiling for disease prediction, diagnosis, or prognosis, but underscores the necessity of care in study design and analysis to account for inter-individual differences and confounding signals.

Fatal Cardiac Arrhythmia and Long-QT Syndrome in a New Form of Congenital Generalized Lipodystrophy with Muscle Rippling (CGL4) Due to PTRF-CAVIN Mutations

2010-03-12T08:00:00Z

Author Summary

Patients with generalized lipodystrophy have a marked lack of body fat. Several gene defects have been described that impede fat synthesis and maturation of fat cells. Here we report on mutations in a novel gene, called PTRF-CAVIN, causing congenital generalized lipodystrophy type 4 (CGL4) that is additionally associated with muscle disease. Patients' muscles are large but weak and show an involuntary, rolling contraction pattern called “rippling.” Further symptoms comprise life-threatening cardiac arrhythmias and a disorder of bone formation. We searched for shared segments in the genome of seven patients and found the responsible gene, called PTRF-CAVIN, on chromosome 17. This gene is crucial for caveolae (latin for “small caves”) formation. These small indentations of the cell membrane are found on the surface of muscle, bone, fat, and immune cells and facilitate cell-to-cell communication and the absorption of substances from the extracellular space. Patients lack more than 97% of caveolae and artificial insertion of the correct gene into patient skin cells led to the reappearance of caveolae. As cardiac arrhythmia is a severe and potentially life-threatening condition, patients with CGL4 should be closely monitored by ECG and, if necessary, fitted with an implanted pacemaker and cardioverter defibrillator (ICD) device.

Identification of the Regulatory Logic Controlling Salmonella Pathoadaptation by the SsrA-SsrB Two-Component System

2010-03-12T08:00:00Z

Author Summary

All organisms have a means to control gene expression ensuring correct spatiotemporal deployment of gene products. In bacteria, gene control presents a challenge because one species can reside in multiple niches, requiring them to coordinate gene expression with environmental sensing. Also, widespread acquisition of DNA by horizontal gene transfer demands a mechanism to integrate new genes into existing regulatory circuitry. The environmental awareness issue can be controlled using two-component regulatory systems that connect environmental cues to transcription factor activation, whereas the integration problem can be resolved using DNA regulatory evolution to create new regulatory connections between genes. The evolutionary significance of regulatory evolution for host adaptation is not fully known. We studied the convergence of environmental sensing and genetic networks by examining how the Salmonella enterica SsrA-SsrB two-component system, activated in response to host cues, has integrated ancestral and acquired genes into a common regulon. We identified a palindrome as the major element apportioning SsrB on the chromosome. SsrB binding sites have been selected to co-regulate a gene program involved in pathogenic adaptation of Salmonella to its host. In addition, our results indicate that promoter architecture emerging from SsrB-dependent regulatory evolution may support both mutualistic and parasitic bacteria-host relationships.

Drosophila Xpd Regulates Cdk7 Localization, Mitotic Kinase Activity, Spindle Dynamics, and Chromosome Segregation

2010-03-12T08:00:00Z

Author Summary

Mutations in human xpd cause three different syndromes—XP (xeroderma pigmentosum), TTD (trichothiodystrophy), and CS (Cockayne syndrome)—and various different phenotypes, such as sun-induced hyperpigmentation of the skin, cutaneous abnormalities, neuronal degeneration, and developmental retardation. In addition, while some mutations cause a highly elevated cancer risk, others do not. The multitask protein Xpd functions in transcription, nucleotide excision repair (NER), and in cell cycle regulation. In a situation where transcription is not required and NER not induced, we specifically analyzed the cell cycle function of Xpd in Drosophila. In this situation Xpd locally controls the dynamic localization of Cdk7, the catalytic subunit of the Cdk activating kinase (CAK) to and away from its cellular targets, thereby regulating mitotic kinase activity and mitotic exit. Xpd also controls spindle dynamics to prevent formation of multipolar and promiscuous spindles and aneuploidy. Through multitask proteins like Xpd and Cdk7 cells regulate different cellular pathways in a coordinated fashion. In addition to the basic research relevance, the newly gained knowledge about the cell cycle function of Xpd and its control of spindle dynamics is also relevant for human xpd patients because it shows a possible pathway that could lead to highly increased cancer risk and neurological defects.

Bias and Evolution of the Mutationally Accessible Phenotypic Space in a Developmental System

2010-03-12T08:00:00Z

Author Summary

Random mutation does not generate random phenotypic variation because genetic and developmental architecture may constrain and bias the mutationally inducible phenotypic spectrum. Understanding such biases in the introduction of phenotypic variation is thus essential to reveal which phenotypes can ultimately be explored and selected through evolution. Here we used lines which had accumulated spontaneous random mutation over 250 generations starting from four distinct wild isolates of the nematode species C. briggsae and C. elegans, to study how a developmental system—vulval cell fate patterning—responds to mutational perturbations. We show that developmental defects and variants increase upon mutation accumulation in lines derived from all four isolates. However, some mutationally induced phenotypic variants occur more frequently than others, and the degree and spectrum of developmental variation further differed between isolates. These results illustrate how the phenotypic spectrum induced by random mutation can be biased due to both developmental system features and variation in the genetic background. Moreover, the mutationally most sensitive phenotypic characters are the ones that show most evolutionary variation among closely related species. These observations show how random mutation translates into a biased, limited range of phenotypes—a phenomenon likely impacting possible trajectories of phenotypic evolution.

Arginylation-Dependent Neural Crest Cell Migration Is Essential for Mouse Development

2010-03-12T08:00:00Z

Author Summary

Formation of many organs during development depends on the coordinated migration of individual cells and cell layers throughout the embryo. The majority of migrating cells originate from the neural crest lineage that gives rise to peripheral neurons, ganglia, pigment cells, and craniofacial structures, as well as parts of other organs in the body. Recent studies have implicated arginylation—a poorly understood protein modification—in the regulation of basic mechanisms that underlie cell migration. Here we test the role of arginylation in neural crest cell migration during mouse development by constructing and examining a mouse model with arginylation-deficient neural crest cells. We find that these mice die at or soon after birth and exhibit severe defects in the development of distinct neural crest-derived structures. Our findings uncover a previously unknown mechanism of the regulation of neural crest cell migration during development, and shed light on general principles of neural crest migration in vivo.

MiR-218 Inhibits Invasion and Metastasis of Gastric Cancer by Targeting the Robo1 Receptor

2010-03-12T08:00:00Z

Author Summary

MicroRNAs have been identified as playing important roles in tumor metastasis, but their impact on GC metastasis has been poorly explored. We have discovered miR-218, which functions as a suppressor of tumor metastasis and is correlated with clinical stage, lymph node metastasis, and prognosis in patients with GC. Our results show that miR-218 is part of a regulatory circuit involving the Slit-Robo1 pathway. In metastatic tumor cells, miR-218 was suppressed along with Slit3, one of its host genes. Meanwhile, Robo1, one of several Slit receptors, is upregulated in response to the decrease in miR-218, which in turn induced a reactive upregulation of the Slit-Robo1 pathway through an interaction with Slit2, thus facilitating tumor cell migration and invasion. Such findings not only provide new insights into the metastatic mechanisms in GC but also provide evidence for a novel miRNA–mediated regulatory mode of receptor signaling.

HP1 Recruitment in the Absence of Argonaute Proteins in Drosophila

2010-03-12T08:00:00Z

Author Summary

One role for silent heterochromatin is to preserve the integrity of the genome by stabilizing regions rich in repetitive sequence and mobile elements. Compaction of repetitive sequences by heterochromatin is needed to prevent genome rearrangement and loss of genetic material. Furthermore, uncontrolled movement of mobile elements throughout the genome can result in deleterious mutations. In fission yeast, one important mechanism of heterochromatin establishment occurs through RNA interference, an RNA–dependent gene silencing process. However, it is unclear whether a direct role for RNA silencing in heterochromatin formation is conserved throughout evolution. In the fruit fly, Drosophila melanogaster, which harbors multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, previous studies have suggested the involvement of the endogenous small interfering RNA (endo-siRNA) and Piwi-interacting RNA (piRNA) pathways in heterochromatin formation. These small RNA silencing pathways suppress the expression of mobile elements in the soma or in both somatic and germline tissues, respectively. Utilizing complementary genetic and biochemical approaches, we monitored the heterochromatin state at discrete genomic locations from which both types of these small RNAs originate in endo-siRNA or piRNA pathway mutants. Our results indicate that heterochromatin can form independently of these two small RNA silencing pathways.

In the Tradition of Science: An Interview with Victor Ambros

2010-03-05T08:00:00Z

Role of RecA and the SOS Response in Thymineless Death in Escherichia coli

2010-03-05T08:00:00Z

Author Summary

A long-standing enigma in the fields of DNA repair and cancer chemotherapy is why it is that cells starved of the base thymine die rapidly. This process, called thymineless death (TLD), is conserved in bacterial, yeast, and human cells and is the mode of action of important cancer chemotherapeutic drugs. Tumors that become resistant to those drugs have ceased to die from TLD. Despite its ubiquity, importance, and having been studied for more than 50 years, the mechanism(s) of TLD remained elusive. Here we show that a large fraction of TLD requires RecA, the central protein in homologous recombinational (HR) DNA repair, and activation of the bacterial DNA–damage (or SOS) response, which RecA controls. We find that of the 40 or so proteins upregulated during an SOS response, SulA, an inhibitor of cell division, accounts for most of how SOS–activation causes TLD. In cells undergoing TLD, we observe blocked replication of the E. coli chromosome followed by loss of DNA near the replication origin then terminus. This implies that much of TLD results from an irreversible cell-cycle checkpoint that blocks cell division when single-stranded DNA (the SOS–inducing signal) accumulates and that the rest results from DNA destruction, models for which are presented.

Rapid Assessment of Genetic Ancestry in Populations of Unknown Origin by Genome-Wide Genotyping of Pooled Samples

2010-03-05T08:00:00Z

Author Summary

Many association studies have been published looking for genetic variants contributing to a variety of human traits such as obesity, diabetes, and height. Because the frequency of genetic variants can differ across populations, it is important to have estimates of genetic ancestry in the individuals being studied. In this study, we were able to measure genetic ancestry in populations of mixed ancestry by genotyping pooled, rather than individual, DNA samples. This represents a rapid and inexpensive means for modeling genetic ancestry and thus could facilitate future association or population-genetic studies in populations of unknown ancestry for which whole-genome data do not already exist.

Association of the OCA2 Polymorphism His615Arg with Melanin Content in East Asian Populations: Further Evidence of Convergent Evolution of Skin Pigmentation

2010-03-05T08:00:00Z

Author Summary

Our knowledge of the genetic basis of normal pigmentation variation in human populations is quite incomplete. Recent studies have identified some of the genes responsible for the reduction in melanin content in European populations, but this is not the case for other population groups, such as East Asians. Here, we report that a genetic variant located within the gene OCA2 (rs1800414) is associated with skin pigmentation in two samples of East Asian ancestry. The allele associated with lower melanin levels is found at high frequencies in East Asian populations, but is absent or at very low frequencies in other population groups. This is one of the first reports of association of genetic markers with quantitative measures of pigmentation in East Asian populations and it confirms previous evidence indicating that evolution towards light skin occurred, at least in part, independently in Europe and East Asia. The OCA2 gene has been under positive selection in Europe and East Asia, but different alleles have been selected in each region.

Derepression of the Plant Chromovirus LORE1 Induces Germline Transposition in Regenerated Plants

2010-03-05T08:00:00Z

Author Summary

In contrast to animals, where germline differentiation initiates early in embryogenesis, germline differentiation in plants starts in the adult phase during reproductive development. Transpositions of transposable elements in both somatic and gametic cells can be transmitted to the next generation. As a result, plant genomes may contain transposable elements exhibiting a variety of tissue-specific activities. Thus far, the spatio-temporal activity of LTR retrotransposons, the most abundant class of transposable elements in plants, has not been well characterized. Here, we report a detailed analysis of the spatio-temporal transposition pattern of a plant LTR retrotransposon in the endogenous system. Using the model legume Lotus japonicus, we found that LORE1a, a member of the chromovirus LORE1 family that belongs to the Gypsy superfamily, was epigenetically de-repressed via tissue culture. Activation was stochastic and derepression was maintained in regenerated plants. This feature made it possible to trace the original spatio-temporal activity of the retrotransposon in the intact plants. We determined that the plant chromovirus retrotransposes mainly in the male germline, without obvious insertional preferences for chromosomal regions. This finding suggests that the tissue specificity of transposable elements should be taken into account when considering their impact on the host genome dynamics and evolution.

KRAB–Zinc Finger Proteins and KAP1 Can Mediate Long-Range Transcriptional Repression through Heterochromatin Spreading

2010-03-05T08:00:00Z

Author Summary

The regulation of gene activity by transcription factors is crucial to the function of all cells. Here, we studied the mechanisms of action of the largest family of gene regulators encoded by the human genome, the so-called KRAB–containing zinc finger proteins (KRAB–ZFPs), which in concert with their universal cofactor KAP1 act as transcriptional repressors. For this, we used two parallel approaches. First, by targeting an ectopic KRAB domain to hundreds of different genes, we found that KRAB/KAP1 can repress promoters located several tens of kilobases from the repressor DNA docking site. We further could show that KRAB induces such long-range effects by mediating the spread of repressive chromatin marks along the body of the gene, resulting in a block of transcriptional initiation at the promoter. In a second set of experiments, we analyzed an endogenous KRAB–ZFP gene cluster, where we could also document KAP1–dependent heterochromatin spreading and transcriptional repression. Together, these results support a model whereby KRAB–ZFPs and KAP1 can mediate long-range transcriptional repression through the spread of silencing chromatin marks. This study thus provides insight into KRAB/KAP1–induced gene regulation at KRAB–ZFP gene clusters, and will further help interpret genome-wide studies of KRAB–ZFPs and KAP1 DNA binding patterns.

Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human

2010-03-05T08:00:00Z

Author Summary

Congenital ocular malformations affecting the optic nerve are an important cause of childhood blindness. The papillorenal syndrome (PRS) is an autosomal dominant disorder that causes congenital optic nerve and kidney abnormalities, which may result in legal blindness and renal failure, respectively. Many cases of PRS are caused by mutations in the paired-box transcription factor PAX2. In this paper, we describe a novel mouse model of this human disease caused by a missense mutation in the Pax2 gene at the same position of one of the few disease-causing missense mutations in humans. We characterize the ocular and non-ocular phenotypes of this mouse and model the effect that murine and human Pax2/PAX2 mutations have on protein structure. We also experimentally test the effect these missense mutations have on protein localization, transactivation, and DNA binding, concluding that all three reduce steady-state levels of protein in vitro and (in p.T74A) in vivo by reducing protein stability. This work will help us better understand the pathophysiology of PRS and to dissect the molecular interactions important in normal PAX2 function.

Mislocalization of XPF-ERCC1 Nuclease Contributes to Reduced DNA Repair in XP-F Patients

2010-03-05T08:00:00Z

Author Summary

XPF-ERCC1 is a nuclease that plays a critical role in DNA repair. Mutations in XPF are linked to xeroderma pigmentosum, characterized by sun sensitivity, high incidence of skin cancer, and neurodegeneration, or XFE progeroid syndrome, a disease of accelerated aging. Herein we report the unexpected finding that mutations in XPF cause mislocalization of XPF-ERCC1 to the cytoplasm. Recombinant mutant XPF-ERCC1 derived from XP– and XFE–causing alleles are catalytically active and if delivered to the nucleus of cells restore DNA repair. This demonstrates that protein mislocalization contributes to defective DNA repair and disease arising as a consequence of mutations in XPF. It also illustrates a novel mechanism of regulating a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1 to enhance or inhibit repair and to prevent cancer or tumor resistance to chemotherapy, respectively.

PPS, a Large Multidomain Protein, Functions with Sex-Lethal to Regulate Alternative Splicing in Drosophila

2010-03-05T08:00:00Z

Author Summary

In Drosophila the sex-specific ON/OFF regulation of Sex-lethal (Sxl) is controlled by an autoregulatory splicing mechanism that depends on the SXL protein interacting with general splicing factors. Here we identify PPS as a novel component of the machinery required for Sxl splicing autoregulation by showing that the lack of pps function interferes with Sxl expression and that the PPS protein is physically linked to the Sxl pre–mRNA, the SXL protein and components of the general splicing machinery. PPS, however, stands apart from all other proteins known to control Sxl splicing because it is not a general splicing factor. Furthermore, PPS has a distinct pattern of accumulation along the Sxl transcription unit that suggests PPS is loaded onto the RNA at the promoter. Together with the observation that the PPS protein contains four signature motifs typically found in proteins that function in transcriptional regulation, our data suggest that linking transcription to splicing regulation is important for controlling Sxl expression. This idea is especially intriguing because it indicates that the coupling of transcription and splicing seen in vitro and in cell culture studies is likely to be pertinent to developmentally controlled patterns of gene expression in the living animal.

PLoS Genetics Issue Image | Vol. 6(2) February 2010

2010-02-26T08:00:00Z

A juvenile stickleback head stained red for developing bone and blue for forming cartilage.

In this issue of PLoS Genetics, Hohenlohe et al. report the first high-density genome scan of stickleback fish to identify signatures of selection. Oceanic stickleback have repeatedly formed freshwater populations that have evolved rapidly in many characters, including the head and jaws. Previous work demonstrated that the same genes were behind parallel phenotypic changes in independent populations. Hohenlohe et al. show that this is a general pattern in stickleback evolution, with many genomic regions exhibiting parallel signatures in independently derived freshwater populations. These results show that parallel phenotypic evolution may often be caused by extensive parallel genomic changes.

Image Credit: Mark Currey (Center for Ecology and Evolutionary Biology, University of Oregon, USA)

Bacterial Genes in the Aphid Genome: Absence of Functional Gene Transfer from Buchnera to Its Host

2010-02-26T08:00:00Z

Author Summary

Bacterial lineages have repeatedly evolved intimate symbioses with eukaryotic hosts, the most famous cases being those of the cell organelles, mitochondria, and plastids. Symbiont genomes typically lose many ancestral genes, raising the question of how they function with so few genes. In organelles, part of the answer involves gene transfer to the host genome, allowing maintenance of essential functions. So far, the extent of gene transfer to hosts has not been assessed for other cases of intimate, obligate symbiosis. Aphids harbor an ancient coevolved intracellular symbiont, called Buchnera. We used the newly available sequence of the pea aphid genome to conduct an exhaustive computational search for genes of bacterial ancestry. We found that no functional genes have been transferred from Buchnera, ruling out such transfer as a driving force in genome reduction in this symbiont. However, the aphid genome does contain eight transcribed genes of apparent bacterial origin, some of which have been duplicated after transfer. Based on their expression patterns, most of these appear to function specifically in the aphid-Buchnera symbiosis, presenting the possibility that the maintenance of obligate intracellular symbioses can be affected by the acquisition and duplication of genes transferred from unrelated bacterial lineages.

Cheating by Exploitation of Developmental Prestalk Patterning in Dictyostelium discoideum

2010-02-26T08:00:00Z

Author Summary

Cooperative systems are susceptible to exploitation by cheaters who enjoy the benefits of cooperation without paying the costs. Such conflict is seen in biological systems at every level from individual genes within a cell to individuals within societies. The social amoebae Dictyostelium discoideum have a unique cooperative system in which large numbers of individual cells aggregate to form fruiting bodies with reproductive spores, and dead stalk cells that may help the survival and dispersal of the spores. Fruiting bodies can contain several genotypes, and hence can be exploited by cheater cells that preferentially form spores without contributing fairly to the stalk. We have studied a mutant, cheater C (chtC), which is defective in forming certain stalk cells, but is still able to form fruiting bodies on its own. However, when wild-type cells are mixed with chtC cells, the wild-type cells compensate for the stalk-forming defect of chtC and form more of the stalk cells. In that way, chtC cells cheat by taking advantage of developmental processes that normally regulate cell-type proportions. This study shows that existing mechanisms of developmental regulation can be exploited by cheater mutants, and the social amoebae offer a good system to study such mechanisms.

Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

2010-02-26T08:00:00Z

Author Summary

Humans utilize at least two major pathways to repair DNA double-strand breaks (DSBs): homologous recombination (HR) and non-homologous end joining (NHEJ), and there are at least two genetically discrete sub-pathways of NHEJ: classical-NHEJ (C-NHEJ) and alternative-NHEJ (A-NHEJ). Since the products generated by each of these three repair (sub)pathways differ substantially from one another, it is biologically critical that certain DSBs are repaired by certain DSB repair pathways. How this pathway choice is made in human cells was unclear. In this study, knockout human cell lines that are defective in core C-NHEJ factors were generated. These cell lines are by-and-large extremely deficient in DSB repair, proving that C-NHEJ is the major DSB repair pathway in human cells. Unexpectedly, cell lines reduced for the C-NHEJ factors Ku70 or Ku86, carried out proficient DSB repair because of hyperactive A-NHEJ. In published work we have also demonstrated that Ku suppresses HR throughout the genome and at telomeres. Collectively, these data imply that Ku ensures that C-NHEJ is the major DSB repair pathway by two mechanisms: i) enabling C-NHEJ and ii) by actively suppressing HR and A-NHEJ. Thus, Ku is the critical regulator of pathway choice in human somatic cells.

Genome-Wide Association Study Reveals Multiple Loci Associated with Primary Tooth Development during Infancy

2010-02-26T08:00:00Z

Author Summary

Genome-wide association studies have been used to identify genetic variants conferring susceptibility to diseases, intermediate phenotypes, and physiological traits such as height, hair color, and age at menarche. Here we analyze the NFBC1966 and ALSPAC birth cohorts to investigate the genetic determinants of a key developmental process: primary tooth development. The prospective nature of our studies allows us to exploit accurate measurements of age at first tooth eruption and number of teeth at one year, and also provides the opportunity to assess whether genetic variants affecting these traits are associated with dental problems later in the life course. Of the genes that we find to be associated with primary tooth development, several have established roles in tooth development and growth, and almost half have proposed links with the development of cancer. We find that one of the variants is also associated with occlusion defects requiring orthodontic treatment later in life. Our findings should provide a strong foundation for the study of the genetic architecture of tooth development, which as well as its relevance to medicine and dentistry, may have implications in evolutionary biology since teeth represent important markers of evolution.

Molecular Evolution and Functional Characterization of Drosophila Insulin-Like Peptides

2010-02-26T08:00:00Z

Author Summary

The insulin/IGF signalling (IIS) pathway plays key roles in growth, metabolism, reproduction, and longevity in animals as diverse as flies and mammals. Most multicellular animals contain multiple IIS ligands, including 7 in the fruit fly Drosophila melanogaster (DILP1-7), implying that the diverse functions of IIS could in part be mediated by the functional diversification of the ligands. Although Drosophila is a prime model organism to study IIS, knowledge about the function of individual DILPs is still very limited due to the lack of gene-specific mutants. Therefore, we generated mutants for all 7 dilp genes and systematically analyzed their phenotypes. We show that loss of DILP2 extends lifespan and describe a novel role for DILP6 in growth control. Furthermore, we show that DILPs are evolutionary conserved and can act redundantly, supporting the hypothesis that functional redundancy itself can be of evolutionary advantage. We also describe a specific interaction between IIS and the endosymbiontic bacterium Wolbachia in lifespan regulation. This finding has implications for all longevity studies using IIS mutants in flies and offers the opportunity to study IIS as a mechanism involved in host/symbiont interactions. Finally, we show that DILPs mediate the response of lifespan and fecundity to dietary restriction.

Cdk1 Targets Srs2 to Complete Synthesis-Dependent Strand Annealing and to Promote Recombinational Repair

2010-02-26T08:00:00Z

Author Summary

Broken DNA molecules can be repaired by copying a homologous DNA sequence located elsewhere in the genome. This process, called homologous recombination, needs to be carefully regulated, because unwanted DNA exchanges can lead to genome rearrangements and cell death. Cdk1 kinase is required for cell cycle progression and phosphorylates DNA repair factors, such as Srs2, a protein that can both translocate on single-stranded DNA and open the two strands of DNA double helix. DNA translocation activity of Srs2 is crucial to prevent unwanted recombination, while DNA unwinding activity might be important to promote recombination. In this study, we used two srs2 mutants that constitutively express the unphosphorylated or Cdk1-dependent phosphorylated Srs2 protein isoforms. We found that Srs2 performs genetically distinct functions in preventing or promoting homologous recombination. Cdk1 targets Srs2 to promote accurate repair of double-stranded DNA breaks, but is not essential for the removal of toxic recombination intermediates assembled at single-stranded DNA breaks. Further, Cdk1 counteracts sumoylation of Srs2, which is responsible for recombination defects due to the lack of Srs2 phosphorylation. In summary, Cdk1-dependent Srs2 phosphorylation prevents its unscheduled sumoylation and targets the helicase to promote accurate homologous recombinational repair.

Structure, Function, and Evolution of the Thiomonas spp. Genome

2010-02-26T08:00:00Z

Author Summary

Recent advances in the field of arsenic microbial metabolism have revealed that bacteria colonize a large panel of highly contaminated environments. Belonging to the order of Burkholderiales, Thiomonas strains are ubiquitous in arsenic-contaminated environments. The genome of one of them, i.e. Thiomonas sp. 3As, was deciphered and compared to the genome of several other Thiomonas strains. We found that their flexible gene pool evolved to allow both the surviving and growth in their peculiar environment. In particular, the acquisition by strains of the same species of different genomic islands conferred heavy metal resistance and metabolic idiosyncrasies. Our comparative genomic analyses suggest that the natural environment influences the genomic evolution of these bacteria. Importantly, these results highlight the genomic variability that may exist inside a taxonomic group, enlarging the concept of bacterial species.

Allelic Exchange of Pheromones and Their Receptors Reprograms Sexual Identity in Cryptococcus neoformans

2010-02-26T08:00:00Z

Author Summary

All organisms that undergo sexual reproduction carry out specific mechanisms to establish different sexes. In fungi, sexual identity is typically determined by components housed within specialized regions of chromosomes known as mating-type (MAT) loci. MAT–encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The two mating types of Cryptococcus neoformans (a and α) are specified by an extremely unusual MAT locus. The unique architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created an α strain where the pheromones and pheromone receptor were replaced with the analogous genes from an a strain. We discovered that the resulting strain (α^a) now behaves as if it is an a. It senses and responds to α cells, mates with α cells, and no longer exhibits other α-specific behaviors. Our data show that replacement of two and only two genes completely alters the sexual identity of α cells, establishing pheromones and their receptors as the determinants of sexual identity in C. neoformans.

Nucleoporins and Transcription: New Connections, New Questions

2010-02-26T08:00:00Z

Population Genomics of Parallel Adaptation in Threespine Stickleback using Sequenced RAD Tags

2010-02-26T08:00:00Z

Author Summary

Oceanic threespine stickleback have invaded and adapted to freshwater habitats countless times across the northern hemisphere. These freshwater populations have often evolved in similar ways from the ancestral marine stock from which they independently derived. With the exception of a few identified genes, the genetic basis of this remarkable parallel adaptation is unclear. Here we show that the parallel phenotypic evolution is matched by parallel patterns of nucleotide diversity and population differentiation across the genome. We used a novel high-throughput sequence-based genotyping approach to produce the first high density genome-wide scans of threespine stickleback populations and identified several genomic regions indicative of both divergent and balancing selection. Some of these regions have been associated previously with traits important for freshwater adaptation, but others were previously unidentified. Within these genomic regions we identified candidate genes, laying the foundation for further genetic and functional study of key pathways. This research illustrates the complementary nature of laboratory mapping, functional genetics, and population genomics.

Cdk2 Is Required for p53-Independent G2/M Checkpoint Control

2010-02-26T08:00:00Z

Author Summary

Metazoan cells contain multiple Cdks that regulate cell cycle progression. Recent studies have shown that mouse cells can grow normally with just Cdk1. The roles of the non-essential Cdks remain a fundamental question. In this study, we describe the generation and detailed characterization of CDK2-knockout human somatic cells. Our study demonstrates that Cdk2 is required for robust DNA damage checkpoint signaling. Loss of Cdk2 caused a marked deficiency in the G₂/M arrest—a basic response to DNA damage—in cells that were also nullizygous for P53. We propose that the multiple Cdks present in metazoan cells provide a mechanism by which the cell cycle can be efficiently halted after DNA damage. Significantly, this study reveals a heretofore unrecognized dependence for Cdk2 in p53-deficient cancer cells.

The Genetic Interpretation of Area under the ROC Curve in Genomic Profiling

2010-02-26T08:00:00Z

Author Summary

Genome-wide association studies in human populations have facilitated the creation of genomic profiles that combine the effects of many associated genetic variants to predict risk of disease. However, genomic profiles are inherently constrained in their ability to classify diseased from non-diseased individuals dictated by the genetic epidemiology of the disease. In this paper, we use a genetic interpretation to provide insight into the constraints on genomic profiles for risk prediction. We provide a strategy to estimate proportion of genetic variance explained on the liability scale from estimates of AUC, disease prevalence, and heritability available as an online calculator.

A Genome-Wide Association Study Identifies Susceptibility Variants for Type 2 Diabetes in Han Chinese

2010-02-19T08:00:00Z

Author Summary

Type 2 diabetes (T2D) is a complex disease that involves many genes and environmental factors. Genome-wide and candidate-gene association studies have thus far identified at least 19 regions containing genes that may confer a risk for T2D. However, most of these studies were conducted with patients of European descent. We studied Chinese patients with T2D and identified two genes, PTPRD and SRR, that were not previously known to be involved in diabetes and are involved in biological pathways different from those implicated in T2D by previous association reports. PTPRD is a protein tyrosine phosphatase and may affect insulin signaling on its target cells. SRR encodes a serine racemase that synthesizes D-serine from L-serine. Both D-serine (coagonist) and the neurotransmitter glutamate bind to NMDA receptors and trigger excitatory neurotransmission in the brain. Glutamate signaling also regulates insulin and glucagon secretion in pancreatic islets. Thus, SRR and D-serine, in addition to regulating insulin and glucagon secretion, may play a role in the etiology of T2D. Our study suggests that, in different patient populations, different genes may confer risks for diabetes. Our findings may lead to a better understanding of the molecular pathogenesis of T2D.

Genome-Wide Identification of Binding Sites Defines Distinct Functions for Caenorhabditis elegans PHA-4/FOXA in Development and Environmental Response

2010-02-19T08:00:00Z

Author Summary

The C. elegans transcription factor PHA-4 is a member of the highly conserved FOXA family of transcription factors. These factors act as master regulators of organ development by controlling how genes are turned off and on as tissues are formed. Additionally they regulate genes in response to nutrient levels and control both longevity and survival of the organism. However, the extent to which these factors control similar or distinct gene targets for each of these functions is unknown. For this reason, we have used the technique of chromatin immunoprecipitation followed by deep sequencing (ChIP–Seq), to define the target binding sites of PHA-4 on a genome-wide scale, when it is either functioning as an organ identity regulator or in response to environmental stress. Our data clearly demonstrate distinct sets of biologically relevant target genes for the transcription factor PHA-4 under these two different conditions. Not only have we defined PHA-4 targets, but we established an experimental ChIP–Seq pipeline to facilitate the identification of binding sites for many transcription factors in the future.

Genome-Wide Identification of Susceptibility Alleles for Viral Infections through a Population Genetics Approach

2010-02-19T08:00:00Z

Author Summary

Viruses have represented a constant threat to human communities throughout their history, therefore, human genes involved in anti-viral response can be thought of as targets of virus-driven selective pressure. Here we utilized the marks left by selection to identify viral infection-associated allelic variants. We analyzed more than 660,000 single nucleotide polymorphisms (SNPs) genotyped in 52 human populations, and we used virus diversity (the number of different viruses in a geographic region) to measure virus-driven selective pressure. Results showed that genes involved in immune response and in the biosynthesis of glycan structures functioning as viral receptors display more variants associated with virus diversity than expected by chance. The same holds true for genes encoding proteins that directly interact with viral components. Genome-wide analysis identified 441 variants, mapping to 139 human genes, significantly associated with virus-diversity. We analyzed the functional relationships among genes subjected to virus-driven selective pressure and identified a complex interaction network enriched in viral products-interacting proteins. Therefore, we describe a novel approach for the identification of gene variants that may be involved in the susceptibility to viral infections.

Use of DNA–Damaging Agents and RNA Pooling to Assess Expression Profiles Associated with BRCA1 and BRCA2 Mutation Status in Familial Breast Cancer Patients

2010-02-19T08:00:00Z

Author Summary

A large number of rare sequence variants of unknown clinical significance have been identified in the breast cancer susceptibility genes, BRCA1 and BRCA2. Laboratory methods to identify which of these variants are mutations would have utility for counseling and clinical decision making when identified in patients with a family history of breast cancer. We used DNA–damaging agents to disturb gene expression profiles of cell-lines derived from blood of patients, and we compared patterns from women with BRCA1 and BRCA2 mutations to women familial breast cancer families without such mutations. Using a pooling strategy, which allowed us to compare several treatments at one time, we identified which treatment caused the greatest difference in gene-expression changes between patient groups and used this treatment method for further study. We were able to accurately classify BRCA1 and BRCA2 samples, and our results supported other reported findings that suggested familial breast cancer patients without BRCA1/2 mutations are genetically heterogeneous. We demonstrate a useful strategy to identify treatments that induce gene expression differences associated with BRCA1/2 mutation status. This strategy may aid the development of a molecular-based tool to screen individuals from multi-case breast cancer families for the presence of pathogenic mutations.

Analysis of the Legionella longbeachae Genome and Transcriptome Uncovers Unique Strategies to Cause Legionnaires' Disease

2010-02-19T08:00:00Z

Author Summary

Legionella longbeachae, found in potting soil, and L. pneumophila, present in aquatic environments, are opportunistic human pathogens that cause Legionnaires' disease, a severe and often fatal pneumonia. The analysis and comparison of the genome sequences of four L. longbeachae genomes together with the study of its gene expression program and virulence pattern in different infection models provides important new insight on the organism's lifestyle and virulence strategies. L. longbeachae harbors a unique repertoire of secreted substrates, many of which encode eukaryotic like domains that may help the pathogen to subvert host functions and cause disease. Curiously, L. longbeachae may also be able to interact with plants. Several proteins present mainly in plants and phytopathogenic bacteria and several enzymes that might confer the ability to degrade plant material were identified in its genome. Interestingly, L. longbeachae encodes a chemotaxis system but no flagella, in contrast L. pneumophila encodes flagella but no chemotaxis system. It will be an interesting aspect of future research to understand these peculiarities. Finally, the genome sequence and analysis reported here will aid in understanding how L. longbeachae causes disease and will open new possibilities to develop tools for rapid identification and risk prediction of L. longbeachae infection.

Proteasome Nuclear Activity Affects Chromosome Stability by Controlling the Turnover of Mms22, a Protein Important for DNA Repair

2010-02-19T08:00:00Z

Author Summary

Chromosome Instability (CIN) is a genome phenotype that involves changes in chromosome number or structure, and accounts for most malignancies. In this paper, we describe a screen to identify a set of novel CIN genes and find that proteasomal subunits represent a major functional group. We show that proteasome dysfunction affects CIN by impairing DNA double strand break (DSB) repair. Previous studies speculated that the proteasome is required to degrade one or more components of the DSB repair machinery; however, until now, no such target has been identified. Here we identify the previously described CIN gene MMS22 as a proteasomal target. We found that, as a result of DNA damage, Mms22 is ubiquitinated and recruited to chromatin. Mms22 then undergoes polyubiquitination and subsequent proteasome-mediated degradation. We also provide evidence that the degradation of Mms22 is important for the normal course of DNA repair and for exit from the G₂/M arrest induced by DNA damage. Our results demonstrate for the first time that a DSB repair protein is a proteasome target, linking nuclear proteasomal activity and DSB repair. The mechanism of regulation of Mms22 may serve as a paradigm to understand how these additional proteins are regulated by the proteasome.