PLoS Genetics: New Articles

The Germ Cell Nuclear Proteins hnRNP G-T and RBMY Activate a Testis-Specific Exon

2009-11-06T08:00:00Z

Author Summary

This study investigates tissue-specific alternative splicing, which plays a key role in generating diversity in animal cells. We found a new testis-specific exon in a human homologue of the important Drosophila developmental regulator Groucho, which is activated by germ cell RNA binding proteins. By analyzing splicing control of this exon, we elucidated how variations in the activity and expression of splicing regulators together counterbalance splicing activation, and achieve more tightly regulated physiological splicing patterns. We find that although this new human testis-specific exon is not conserved in mice, it is functionally important in that it encodes a peptide which increases the activity of this developmental regulator as a transcriptional repressor. This study provides new insights into how signalling pathways are evolving in human germ cells and the possible molecular defects that might be occurring in infertile men who have genetic deletions of germ cell-specific RNA binding proteins.

Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3′ Splice Site In Vivo

2009-11-06T08:00:00Z

Author Summary

Eukaryotic genes contain intervening intronic sequences that must be removed from pre-mRNA transcripts by RNA splicing to generate functional messenger RNAs. While studying genes that encode and control a presumptive muscle potassium channel complex in the nematode Caenorhabditis elegans, we found that mutations in two splicing factors, the U2AF large subunit and SF1/BBP suppress the rubberband Unc phenotype caused by a rare missense mutation in the gene unc-93. Mutations affecting the U2AF large subunit caused the recognition of a cryptic 3′ splice site generated by the unc-93 mutation, providing in vivo evidence that the U2AF large subunit can affect splice-site selection. By contrast, an SF1/BBP mutation that suppressed the rubberband Unc phenotype did not cause splicing using this cryptic 3′ splice site. Our genetic studies identified a region of the U2AF large subunit important for its effect on 3′ splice-site choice. Our mutagenesis analysis of in vivo transgene splicing identified a positional effect on weak 3′ splice site selection and nucleotides of the endogenous 3′ splice site important for recognition. The system we have defined should facilitate future in vivo analyses of pre–mRNA splicing.

Functional Evolution of cis-Regulatory Modules at a Homeotic Gene in Drosophila

2009-11-06T08:00:00Z

Author Summary

The fertilized animal embryo is a mass of uniform cells that becomes a complex, segmented, and highly organized structure of differentiated cells through the process of development. This vital process is controlled by networks of developmental genes interacting with each other on the molecular level. Because these genes are crucial for animal development, they are conserved both in function and at the DNA sequence level in related species. We have examined critical DNA sequence modules which regulate genes that pattern the early embryo in different species of the fruit fly. We found that despite rapid evolution of the DNA sequences, the regulatory sequences from one fruit fly species are able to operate when tested in another fruit fly species. Further analysis reveals that there are sequences within these regulatory DNA modules which are conserved across different species and which are critical for regulatory function. These conserved sequences represent critical binding sites for protein transcription factors. These findings have important implications for our understanding of gene regulation during development and evolution across diverse animal species ranging from the fruit fly to humans.

The Sir2-Sum1 Complex Represses Transcription Using Both Promoter-Specific and Long-Range Mechanisms to Regulate Cell Identity and Sexual Cycle in the Yeast Kluyveromyces lactis

2009-11-06T08:00:00Z

Author Summary

Sir2 deacetylases are found in organisms ranging from bacteria to mammals. Sir2 from the yeast Saccharomyces cerevisiae deacetylates histones and is part of the SIR complex that spreads across chromatin to repress gene expression. A related histone deacetylase, Hst1, interacts with a DNA–binding protein, Sum1, to repress genes in a promoter-specific manner. Hst1 and Sir2 are paralogs, arising from a duplication about 100 million years ago. To understand how Sir2 and Hst1 have diverged, as well as to investigate the evolutionary relationship between spreading and non-spreading mechanisms of gene repression, we have characterized the function of a non-duplicated Sir2 from the yeast Kluyveromyces lactis, a species that diverged from Saccharomyces prior to this duplication. We found that KlSir2 is part of both the SIR and SUM1 complexes, indicating that the ancestral Sir2 had both Sir2- and Hst1-like properties. Interestingly, we found that, in K. lactis, the Sir2-Sum1 complex not only uses a promoter-specific mechanism to repress the same sets of genes as S. cerevisiae, it also forms extended chromatin structures to repress gene transcription. Our results illustrate how mechanisms by which regulatory proteins act can change over evolutionary time.

OAZ-t/OAZ3 Is Essential for Rigid Connection of Sperm Tails to Heads in Mouse

2009-11-06T08:00:00Z

Author Summary

Polyamines are essential for cell proliferation and differentiation, but their role in these processes is unknown. Ornithine decarboxylase antizymes (OAZs) are enzymes that control the concentration of polyamines in cells. To elucidate the role of one of these enzymes, OAZ-t, in the regulation of polyamine concentration during sperm formation, we generated mutant mice in which the OAZ-t gene was disrupted. When we observed sperm from the mice lacking a functional Oaz-t gene, we found that the sperm heads separated easily from the tails, indicating that OAZ-t is essential for the formation of a rigid junction between the head and tail during sperm development. Many of the headless tails could continue swimming, but they were unable to participate in the signaling processes required for successful fertilization. However, tailless heads could produce healthy pups when injected into unfertilized eggs. Such a phenotype has not been previously found. The mutant mice evoked rare cases of infertile human patients whose sperm behaves in a proper fashion. Our study underscores the importance of research into the processes of spermatogenesis and fertilization.

E Unibus Plurum: Genomic Analysis of an Experimentally Evolved Polymorphism in Escherichia coli

2009-11-06T08:00:00Z

Authors Summary

Experimental evolution of asexual species has shown that multiple genotypes can arise from a single ancestor and stably coexist (e unibus plurum). Although facilitated by environmental heterogeneity, this phenomenon also occurs in simple, homogeneous environments provisioned with a single limiting nutrient. We sought to discover genetic mechanisms that enabled an E. coli population founded by a single clone to become an interacting community composed of multiple clones. The founder of this population contained mutations that impair regulation of acetate and glycerol metabolism and likely favored the evolution of cross-feeding. Adaptive clones share cis- and trans-regulatory mutations shown elsewhere to enhance fitness under glucose limitation. Certain mutations that distinguish adaptive clones and underlie evolution of specialists were compensatory rather than gain-of-function, and all that we detected resulted in gene expression changes rather than protein structure changes. Evolved clones exhibited both common and clone-specific gene expression changes relative to their common ancestor; the pattern of gene expression in the dominant clone cultured alone differed from the pattern observed when it was cultured with variants feeding on its overflow metabolites. These findings illuminate the roles played by founder genotype, differential gene regulation, and the biotic environment in the adaptive evolution of bacteria.

Localized Plasticity in the Streamlined Genomes of Vinyl Chloride Respiring Dehalococcoides

2009-11-06T08:00:00Z

Author Summary

Dehalococcoides are free-living sediment and subsurface bacteria with remarkably small, streamlined genomes and an unusual degree of niche specialization. These strictly anaerobic bacteria gain metabolic energy exclusively through a novel type of respiration that results in reductive elimination of chlorides from organochlorines, many of which are priority pollutants. In this article, we compare the first complete genome sequences of Dehalococcoides strains that grow via respiration of vinyl chloride (VC), a human carcinogen and abundant groundwater pollutant. Our work provides novel insights into Dehalococcoides chromosome organization and evolution, identifies specific positions in the chromosomes where new genes—like the genes responsible for growth on VC—are integrated, and generates clues how these dechlorinating bacteria adapt to anthropogenic contamination. This information sheds new light on Dehalococcoides biology and ecology, with implications for enhanced bioremediation to protect dwindling drinking water reservoirs.

PLoS Genetics Issue Image | Vol. 5(10) October 2009

2009-10-30T07:00:00Z

Five-week-old juvenile gray mouse lemurs (Microcebus murinus) in a hollow tree in Kirindy Forest, Western Madagascar.

Lemurs are the most distantly related non-human primates. A Research Article by Anne Averdam and colleagues in this issue of PLoS Genetics (see Averdam et al., 10.1371/journal.pgen.1000688) shows that lemurs differ considerably from "higher" primates in receptors of natural killer (NK) cells and their ligands. Instead of KIR or Ly49 genes, lemurs substantially expanded and diversified CD94/NKG2 receptors. Remarkably, this novel system of polymorphic and diverse receptors points to combinatorial diversity as a mechanism to increase the NK cell receptor repertoire of lemurs, a finding that was previously unknown for NK cells.

Image Credit: Manfred Eberle, www.phocus.org (German Primate Center - Leibniz Institute for Primate Research, Germany)

Dissection of the Complex Phenotype in Cuticular Mutants of Arabidopsis Reveals a Role of SERRATE as a Mediator

2009-10-30T07:00:00Z

Author Summary

As the skin of a plant, the epidermis mediates a broad set of protective functions which includes defense against abiotic environmental stresses and pathogens. The majority of its barrier capacity is localized to the outermost cell wall, which is covered by a waxy cuticle. Several distinct cuticular mutants in the model plant Arabidopsis produce a remarkable syndrome that is characterized by ectopic cell adhesion and changes in plant morphology. We used these mutants to study the constitution of the cuticle and the activation of the molecular compensatory mechanisms that are important for adaptation. We examined whole-genome responses in these mutants and used an appropriate statistical procedure to reveal the genes which change their expression. We then applied the same approach to the analysis of hundreds of datasets in repositories. The comparison of gene expression profiles identified the gene SERRATE, which encodes a protein of RNA–processing multi-protein complexes, and further analysis revealed that the syndrome is suppressed in double mutants, as predicted. Our finding suggests that the mechanism which operates to control the integrity of the cuticle involves the regulation of small–RNA signaling.

Progressive GAA·TTC Repeat Expansion in Human Cell Lines

2009-10-30T07:00:00Z

Author Summary

The human genome is comprised of the DNA base sequences used by the cell as a blueprint to direct proper cellular function. Changes in this sequence, known as genomic instability, often interfere with vital cellular functions, resulting in genetic disorders. Repetitive DNA sequences are particularly susceptible to genomic instability. Trinucleotide repeat disorders are caused by three base repeat sequences that increase in size when passed from parent to child and during aging. Trinucleotide repeat expansion results in disease when the size of the repeat sequence increases into the pathogenic size range. Our understanding of the mechanisms responsible for these repeat length changes is incomplete and modeling repeat expansion in human cells has proven difficult. Here, we have developed a unique human cellular model of GAA·TTC trinucleotide repeat expansion, the causative mutation in Friedreich ataxia. Using this model, we characterize GAA·TTC expansion in human cells and identify gene transcription as a key regulator of GAA·TTC repeat expansion. The findings of this study provide novel insight into the mechanisms contributing to trinucleotide repeat expansion in human cells and present new implications for certain therapeutic approaches in Friedreich ataxia.

Acquisition of Aneuploidy Provides Increased Fitness during the Evolution of Antifungal Drug Resistance

2009-10-30T07:00:00Z

Author Summary

C. albicans, the most prevalent human fungal pathogen, acquires resistance to fluconazole by genetic alterations that often include changes in the number of chromosomes or chromosome arms (aneuploidy). Here we demonstrate that chromosomal rearrangements resulting in increased gene dosage are the predominant means of acquired resistance to the antifungal drug fluconazole in replicated experimental populations of C. albicans. A specific aneuploidy, isochromosome 5L, which is composed of two copies of the left arm of Chr5, occurs with high frequency and is detectable soon after fluconazole exposure. The early appearance of aneuploidy in some populations is consistent with a model in which C. albicans becomes more permissive of chromosome rearrangements and segregation defects in the presence of fluconazole. The results presented here indicate that the C. albicans genome is highly plastic and imply that exposure to an antifungal drug induces genome reorganization events, some of which provide a fitness advantage in the presence of drug.

The Key Role of Genomics in Modern Vaccine and Drug Design for Emerging Infectious Diseases

2009-10-26T07:00:00Z

It can be argued that the arrival of the “genomics era” has significantly shifted the paradigm of vaccine and therapeutics development from microbiological to sequence-based approaches. Genome sequences provide a previously unattainable route to investigate the mechanisms that underpin pathogenesis. Genomics, transcriptomics, metabolomics, structural genomics, proteomics, and immunomics are being exploited to perfect the identification of targets, to design new vaccines and drugs, and to predict their effects in patients. Furthermore, human genomics and related studies are providing insights into aspects of host biology that are important in infectious disease. This ever-growing body of genomic data and new genome-based approaches will play a critical role in the future to enable timely development of vaccines and therapeutics to control emerging infectious diseases.

Toward the Use of Genomics to Study Microevolutionary Change in Bacteria

2009-10-26T07:00:00Z

Bacteria evolve rapidly in response to the environment they encounter. Some environmental changes are experienced numerous times by bacteria from the same population, providing an opportunity to dissect the genetic basis of adaptive evolution. Here I discuss two examples in which the patterns of rapid change provide insight into medically important bacterial phenotypes, namely immune escape by Neisseria meningitidis and host specificity of Campylobacter jejuni. Genomic analysis of populations of bacteria from these species holds great promise but requires appropriate concepts and statistical tools.

Meta-Analysis of the INSIG2 Association with Obesity Including 74,345 Individuals: Does Heterogeneity of Estimates Relate to Study Design?

2009-10-23T07:00:00Z

Author Summary

A polymorphism of the INSIG2 gene was identified as being associated with obesity in one of the first genome-wide association studies. However, this association has since then been highly debated upon inconsistent subsequent reports. We collected association information from 34 studies including a total of 74,000 participants. In a meta-analysis of the 27 studies including 66,000 Caucasian adults, we found no overall association of this polymorphism rs7566605 with obesity, comparing subjects with a body-mass-index (BMI)≥30 kg/m² with normal BMI subjects (BMI<30 kg/m²). Our data suggested an association of this polymorphism with extreme obesity (e.g., BMI≥37.5 kg/m²) compared to normal controls. Such an association with extreme obesity might induce heterogeneous effects from different study designs depending on the proportion of extreme obesity included by the design. However, further studies would be required to substantiate this finding. The importance of study design might be under-recognized in gene discovery and association replication so far.

Inferring the Joint Demographic History of Multiple Populations from Multidimensional SNP Frequency Data

2009-10-23T07:00:00Z

Author Summary

The demographic history of our species is reflected in patterns of genetic variation within and among populations. We developed an efficient method for calculating the expected distribution of genetic variation, given a demographic model including such events as population size changes, population splits and joins, and migration. We applied our approach to publicly available human sequencing data, searching for models that best reproduce the observed patterns. Our joint analysis of data from African, European, and Asian populations yielded new dates for when these populations diverged. In particular, we found that African and Eurasian populations diverged around 100,000 years ago. This is earlier than other genetic studies suggest, because our model includes the effects of migration, which we found to be important for reproducing observed patterns of variation in the data. We also analyzed data from European, Asian, and Mexican populations to model the peopling of the Americas. Here, we find no evidence for recurrent migration after East Asian and Native American populations diverged. Our methods are not limited to studying humans, and we hope that future sequencing projects will offer more insights into the history of both our own species and others.

High-Density SNP Screening of the Major Histocompatibility Complex in Systemic Lupus Erythematosus Demonstrates Strong Evidence for Independent Susceptibility Regions

2009-10-23T07:00:00Z

Author Summary

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and involvement of multiple organ systems. Although the cause of SLE remains unknown, several lines of evidence underscore the importance of genetic factors. As is true for most autoimmune diseases, a substantial genetic contribution to disease risk is conferred by major histocompatibility complex (MHC) gene(s) on chromosome 6. This region of the genome contains a large number of genes that participate in the immune response. However, the full contribution of this genomic region to SLE risk has not yet been defined. In the current study we characterize a large number of SLE patients and family members for approximately 2,000 MHC region variants to identify the specific genes that influence disease risk. Our results, for the first time, implicate four different MHC regions in SLE risk. We provide a list of candidate variants based on biologic and functional considerations that may be causally related to SLE risk and warrant further investigation.

Loss of Yeast Peroxiredoxin Tsa1p Induces Genome Instability through Activation of the DNA Damage Checkpoint and Elevation of dNTP Levels

2009-10-23T07:00:00Z

Author Summary

Peroxiredoxins are a family of antioxidant enzymes highly conserved from yeast to human. Loss of peroxiredoxin in mice can lead to severe anemia and malignant tumors, but the underlying cause is not understood. One way to derive new knowledge of peroxiredoxins is through genetic analysis in yeast. We have shown that loss of peroxiredoxins in yeast is associated with an increase in mutation rates. Here, we demonstrate that this elevation of mutation rates in yeast cells lacking a peroxiredoxin is due to increased production of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Our findings suggest a new model in which compromised antioxidant defense causes accumulation of damaged DNA and activation of the DNA damage checkpoint. For yeast cells to survive DNA damage, dNTP production is increased to facilitate DNA replication, but at the price of high mutation rates. This new model could lead to a better understanding of human diseases including cancer.

Quantifying Adaptive Evolution in the Drosophila Immune System

2009-10-23T07:00:00Z

Author Summary

All organisms are attacked by an ever-changing array of pathogens and parasites, and it is widely supposed that the ensuing host–parasite “arms race” must drive extensive adaptive evolution in genes of the immune system. Here we have taken advantage of new sequencing technologies and analytical approaches to quantify the amount of adaptation that is occurring in immunity genes relative to the rest of the genome. We sampled two species of fruit fly (D. melanogaster and D. simulans) from eight different populations around the world, and sequenced 136 immunity and 287 non-immunity genes from these samples. Based on the differences in the sequences between the two species, and the genetic diversity within each species, we have estimated that natural selection drives twice as much change in immune-related proteins as in proteins with no immune function. Interestingly, the rate of adaptation is also more variable among immunity genes than among other genes in the genome, with a small subset of immunity genes evolving under intense natural selection. We suggest that these genes may represent hotspots of host–parasite coevolution within the genome.

Restricting Dosage Compensation Complex Binding to the X Chromosomes by H2A.Z/HTZ-1

2009-10-23T07:00:00Z

Author Summary

In organisms where females have two X chromosomes and males only have one, a mechanism called dosage compensation ensures that both sexes receive the same amount of information from their X chromosomes. Disruption of dosage compensation leads to lethality in the affected sex. While the precise mechanisms of dosage compensation differ between organisms, changes to the structure of the X chromosomes are involved in each case. The DNA of all chromosomes is packaged into a complex protein–DNA structure called chromatin. The most basic level of packaging involves wrapping DNA around a group of small proteins called histones. In both mammals and flies, dosage compensation is associated with specific changes to the histones on the dosage compensated X chromosome. Until now, no such change has been associated with dosage compensation in worms. Here we present evidence that the histone variant HTZ-1/H2A.Z plays a role in dosage compensation in the worm. Specifically, we suggest that HTZ-1 functions to ensure that only the X chromosomes, and not the other chromosomes, are subjected to dosage compensation. This suggests that, despite different mechanisms, one common theme of dosage compensation is a change at the level of the histones associated with the chromosomal DNA.

Bruchpilot in Ribbon-Like Axonal Agglomerates, Behavioral Defects, and Early Death in SRPK79D Kinase Mutants of Drosophila

2009-10-23T07:00:00Z

Author Summary

Neurons communicate through release of neurotransmitters at specialized contacts called synapses. Modulation of synaptic transmission likely underlies all higher brain function including feature abstraction, learning and memory, and cognition. The complex molecular machinery that regulates neurotransmitter release has been conserved in evolution but is still incompletely understood. Using the genetic model organism Drosophila, we recently discovered a protein of the presynaptic ribbon (T-bar) that was called Bruchpilot (German for crash pilot) because flies with reduced amounts of this protein cannot fly. We now screened various Drosophila mutants for changes in tissue localization of Bruchpilot and discovered a gene that codes for an enzyme which is similar to mammalian kinases that phosphorylate splicing factors and may co-localize with Bruchpilot at the synapse. Larval nerves of mutants for this gene contain conspicuous accumulations of Bruchpilot that correspond to extensive electron-dense ribbon-like agglomerates surrounded by vesicles. While general axonal transport and basic synaptic transmission at larval nerve-muscle synapses are not affected, adult mutants show reduced life span and impaired flight and walking. The substrate for this kinase and its role in maintaining brain function must now be identified. Its discovery raises important questions about the function of homologous proteins in mammals including humans.

1+1 = 3: A Fusion of 2 Enzymes in the Methionine Salvage Pathway of Tetrahymena thermophila Creates a Trifunctional Enzyme That Catalyzes 3 Steps in the Pathway

2009-10-23T07:00:00Z

Author Summary

Fusion genes, composed of the complete sequence of two or more other genes, are excellent markers of evolution. In addition, fused genes are usually composed of genes with related functions, which makes them useful in inferring function when the function of one of their components is known. We detected a fusion gene in the eukaryotic organism Tetrahymena thermophila that, although composed of only two genes, seems to perform the function of three genes in this organism. To show that this is the case, we expressed the Tetrahymena fused gene in three different yeast strains, each lacking one of these three genes. The Tetrahymena gene was able to rescue the phenotype of all yeast strains, proving that it can perform the functions of the three genes in yeast. Our results highlight another important biochemical characteristic of fusion genes: they can serve as biological shortcuts, allowing a single fusion of two enzymes to functionally replace three independent enzymes.

Mouse HORMAD1 and HORMAD2, Two Conserved Meiotic Chromosomal Proteins, Are Depleted from Synapsed Chromosome Axes with the Help of TRIP13 AAA-ATPase

2009-10-23T07:00:00Z

Author Summary

Generation of haploid gametes in most organisms requires that homologues become connected via crossovers during meiosis. Efficient formation of crossovers depends on HORMA-domain proteins in diverse taxa. These proteins ensure that programmed meiotic DSBs are preferentially repaired from homologues, rather than from sister chromatids. This inter-homologue bias is crucial for homology search and crossovers formation. HORMA-domain proteins have been also implicated in DSB formation, in suppression of synaptonemal complex formation between non-homologous chromosomes, and in the meiotic prophase checkpoint that monitors DSB repair. Despite the importance of HORMA-domain proteins in various organisms, a role for these proteins in mammalian meiosis hasn't been reported. We examined the behaviour of meiotic mouse HORMA-domain proteins—HORMAD1 and HORMAD2—in wild-type and meiotic mutants. HORMAD1/2 preferentially accumulate on unsynapsed chromosome axes. Our data suggest that HORMAD1/2 depletion from chromosomes is a response to synaptonemal complex formation and it that is a conserved process supported by TRIP13/Pch2 AAA-ATPase. Assuming that HORMA-domain functions are conserved in mammals, we speculate that depletion of HORMADs from axes might contribute to the down-regulation of inter-homologue bias and the prophase checkpoint once homology search is completed and synaptonemal complexes form between aligned homologues.

On the Track of DNA Methylation: An Interview with Adrian Bird

2009-10-16T07:00:00Z

Acetylation by the Transcriptional Coactivator Gcn5 Plays a Novel Role in Co-Transcriptional Spliceosome Assembly

2009-10-16T07:00:00Z

Author Summary

Pre-messenger RNA splicing, the removal of non-coding RNA sequences (introns) that interrupt the protein-coding sequence of genes, is required for proper gene expression. While recent studies have revealed that intron recognition begins while the RNA is actively being synthesized by RNA polymerase II, little is known about how the proteins involved in gene transcription and RNA splicing interact to coordinate the two reactions. Here we show that the protein complex SAGA, which allows RNA polymerase II to navigate the three-dimensional structure of packaged DNA by acetylating histone proteins, has an additional role in pre-messenger RNA splicing. Our genetic analysis shows that the SAGA complex has functional interactions with specific components of the splicing machinery. Furthermore, SAGA's acetylation activity, which we find to be targeted toward promoter-bound histones of intron-containing genes, is required for proper recruitment of these components to RNA during active transcription. Our work supports a model whereby SAGA–dependent acetylation facilitates recruitment of the splicing machinery to the pre–mRNA for proper co-transcriptional splicing.

Limiting the Persistence of a Chromosome Break Diminishes Its Mutagenic Potential

2009-10-16T07:00:00Z

Author Summary

A deleterious lesion in DNA is a break of both strands, or a chromosome double-strand break (DSB). DSBs can arise during normal cellular metabolism, but are also a consequence of many forms of cancer therapy. If DSBs are not repaired prior to cell division, entire segments of a chromosome can be lost. Several pathways ensure that DSBs are repaired, though some pathways are prone to causing mutations and/or chromosomal rearrangements, each of which can contribute to cancer development. In the first part of this study, we describe the roles of individual genetic factors in distinct repair pathways of DSBs generated by the I-SceI endonuclease. From these studies, we find that some factors can function in multiple repair pathways. In the second part of this study, we present a method for partially degrading the cohesive DSB overhangs that are generated by I-SceI, which we find facilitates repair products that are not prone to being re-cut by the endonuclease. As a consequence, we have limited the persistence of such breaks, which we find causes a reduction in repair pathways that lead to significant genetic loss. As well, we use this method to characterize the role of individual genetic factors during the repair of non-cohesive DSB ends.

The Elongator Complex Interacts with PCNA and Modulates Transcriptional Silencing and Sensitivity to DNA Damage Agents

2009-10-16T07:00:00Z

Author Summary

During S phase of the cell cycle, not only must DNA sequences be faithfully duplicated, chromatin structures must also be inherited into daughter cells to maintain gene expression states and cell identity. While significant progress has been made in understanding the regulation of DNA replication, how chromatin structures are maintained from one cell division cycle to the next (so-called epigenetic inheritance) is only partially understood. It is believed that the DNA replication-coupled nucleosome assembly process plays an important role in such inheritance as well as maintenance of genome stability. In this process, histone chaperones such as chromatin assembly factor 1 (CAF-1) deposit newly synthesized histones H3–H4, which are acetylated at specific lysine residues, onto replicating DNA in a PCNA dependent reaction. PCNA is a clamp for DNA polymerases and other proteins that are involved in DNA replication and DNA repair. Genetic interactions between lysine acetyltransferase Elp3 and factors involved in DNA replication-coupled nucleosome assembly are described. Elp3 is required for transcriptional silencing and for maintenance of genome stability and binds directly to PCNA. A role for the Elongator complex in response to DNA damage and in maintenance of gene silencing is discussed.

Sequential Use of Transcriptional Profiling, Expression Quantitative Trait Mapping, and Gene Association Implicates MMP20 in Human Kidney Aging

2009-10-16T07:00:00Z

Author Summary

Although family studies have shown that genes play a role in longevity, it has proven difficult to identify the specific genetic variants involved. We developed a sequential transcriptional profiling and eQTL mapping approach to find genes associated with aging in the kidney. First, we used genome-wide transcriptional profiling to determine which genes change expression with age in kidney tissue. Next, we used two methods to determine which of these age-regulated genes contain SNPs that associate with expression level. The allele-specific expression method, which compares the mRNA levels of the two alleles within heterozygous individuals, was more sensitive than the total expression method. We tested the eQTLs for association with kidney aging in two populations. One gene that encodes an extracellular matrix protein, MMP20, significantly associated with kidney aging, providing the first gene association with kidney aging. Our approach of combining both expression and genotype data can be applied to any phenotype of interest to increase the power to find genetic associations.

A Genealogical Interpretation of Principal Components Analysis

2009-10-16T07:00:00Z

Author Summary

Genetic variation in natural populations typically demonstrates structure arising from diverse processes including geographical isolation, founder events, migration, and admixture. One technique commonly used to uncover such structure is principal components analysis, which identifies the primary axes of variation in data and projects the samples onto these axes in a graphically appealing and intuitive manner. However, as the method is non-parametric, it can be hard to relate PCA to underlying process. Here, I show that the underlying genealogical history of the samples can be related directly to the PC projection. The result is useful because it is straightforward to predict the effects of different demographic processes on the sample genealogy. However, the result also reveals the limitations of PCA, in that multiple processes can give the same projections, it is strongly influenced by uneven sampling, and it discards important information in the spatial structure of genetic variation along chromosomes.

The Euchromatic and Heterochromatic Landscapes Are Shaped by Antagonizing Effects of Transcription on H2A.Z Deposition

2009-10-16T07:00:00Z

Author Summary

DNA in living cells is packaged into chromatin by histones and non-histone proteins. This packaging is very dynamic, allowing the controlled access of regulatory proteins such as transcription factors to DNA. Most chromatin is packaged with so-called canonical histones; namely H2A, H2B, H3, and H4. In some regions, however, variant histones replace canonical histones, creating special chromatin regions. Here we show that the variant histone H2A.Z is dynamically recruited to promoter regions where it helps in the recruitment of RNA polymerase II, the enzyme responsible for the first step of gene expression. In addition, we show that H2A.Z also associates randomly in the genome, but these molecules are removed during the passage of RNA polymerase II. In non-transcribed regions, H2A.Z accumulates in large domains called heterochromatin. We propose that a battle between random H2A.Z deposition and RNAPII-dependent H2A.Z eviction shapes the chromatin landscape.

A Novel System of Polymorphic and Diverse NK Cell Receptors in Primates

2009-10-16T07:00:00Z

Author Summary

Most receptors of natural killer (NK) cells interact with highly polymorphic major histocompatibility complex (MHC) class I molecules and thereby regulate the activity of NK cells against infected or malignant target cells. Whereas humans, apes, and Old and New World monkeys use the family of killer cell immunoglobulin-like receptors (KIR) as highly diverse NK cell receptors, this function is performed in rodents by the diverse family of lectin-like receptors Ly49. When did this functional separation occur in evolution? We followed this by investigating lemurs, primates that are distantly related to humans. We show here that lemurs employ the CD94/NKG2 family as their highly diversified NK cell receptors. The CD94/NKG2 receptors also belong to the lectin-like receptor family, but are rather conserved in “higher” primates and rodents. We could further demonstrate that lemurs have a single Ly49 gene like other primates but lack functional KIR genes of the KIR3DL lineage and show major deviations in their MHC class I genomic organisation. Thus, lemurs have evolved a “third way” of polymorphic and diverse NK cell receptors. In addition, the multiplied lemur CD94/NKG2 receptors can be freely combined, thereby forming diverse receptors. This is, therefore, the first description of some combinatorial diversity of NK cell receptors.

A Novel Mechanism of Transposon-Mediated Gene Activation

2009-10-16T07:00:00Z

Author Summary

Transposons are “jumping genes” that can move from one location within a genome to another. Insertion of a transponson changes the DNA sequence and therefore gives rise to mutations that can activate or inactivate gene expression. Here, we demonstrate for the first time that one such transposon, Insertion Sequence 5 (IS5), when positioned upstream of a metabolic operon (glpFK) of E. coli, can activate the otherwise cryptic expression of the operon. This effect is due solely to a short region at the 3′ end of IS5 that harbors a permanent bend and an overlapping nucleoid protein binding site, both of which are required for maximal gene expression. We demonstrate the importance of phasing and conclude that DNA looping probably plays a role. We also show that another operon, the E. coli lactose operon (lacZYA), can be similarly activated by IS5. Although this is the first study to show that unique sequences within a transposon are necessary and sufficient to activate a downstream silent promoter, similar mechanisms of gene activation may occur for other operons.

A Pol V–Mediated Silencing, Independent of RNA–Directed DNA Methylation, Applies to 5S rDNA

2009-10-16T07:00:00Z

Author Summary

In plant genomes, the RNA–directed DNA methylation (RdDM) process induces de novo methylation of cytosines at repeated sequences. The RNA polymerases Pol IV and Pol V are two key components of the RdDM pathway. Pol IV acts with RDR2 (RNA–dependent RNA polymerase 2) and DCL3 (Dicer-Like protein 3) to generate short interfering RNAs (siRNAs). Pol V, in a partnership including AGO4 (Argonaute4) and DRM2 (Domains Rearranged Methyltransferase 2), drives DNA methylation at the targeted sequence. Changes in 5S (ribosomal DNA) rDNA methylation, 5S rDNA chromatin compaction, and 5S siRNA accumulation in Pol IV/V mutants have been reported. However, 5S rDNA arrays were considered together. In the present study, we observed an overexpression of the atypic 5S-210 transcript, restricted to the 5S rDNA array from chomosome 4. This derepression is specific to the Pol V–loss of function (and not to Pol IV) and comes in addition to the RdDM pathway. The Pol V–loss of function induces also the chromatin decondensation of the derepressed 5S locus at chomosome 4. Our results highlight a new role for Pol V which, suprisingly, appears to be Pol IV– and RdDM–independent.

Targeted Induction of Endoplasmic Reticulum Stress Induces Cartilage Pathology

2009-10-16T07:00:00Z

Author Summary

Mutations in genes for extracellular matrix proteins are generally thought to exert their pathogenic effects because of resulting defects in extracellular matrix. However, it is becoming increasingly clear that such mutations can also have significant effects inside the cell due to the induction of ER stress. Mutations in type X collagen cause a dwarfism called metaphyseal chondrodysplasia type Schmid. A gene targeted mouse model expressing mutant type X collagen exhibited an expanded hypertrophic zone of the growth plate and significant increases in cellular ER stress, as noted previously. VEGF expression was disrupted leading to decreases in the rate of vascular invasion. To directly assess the role of elevated ER stress in disease pathogenesis, transgenic mouse lines expressing an exogenous, ER stress–inducing protein (cog mutant of thyroglobulin—Tg^cog) targeted to hypertrophic chondrocytes were generated. Mice expressing Tg^cog protein showed elevated ER stress, an expanded hypertrophic zone, and reduced bone growth demonstrating that elevated ER stress and the resultant UPR is the principal pathogenic mechanism causing this cartilage pathology. It is possible that therapeutic strategies aimed at alleviating ER stress may be beneficial in this and other connective tissue diseases caused by mutant extracellular matrix genes.

Expression Quantitative Trait Loci Are Highly Sensitive to Cellular Differentiation State

2009-10-16T07:00:00Z

Author Summary

Blood cell development from multipotent hematopoietic stem cells to specialized blood cells is accompanied by drastic changes in gene expression for which the triggers remain mostly unknown. Genetical genomics is an approach linking natural genetic variation to gene expression variation, thereby allowing the identification of genomic loci containing gene expression modulators (eQTLs). In this paper, we used a genetical genomics approach to analyze gene expression across four developmentally close blood cell types collected from a large number of genetically different but related mouse strains. We found that, while a significant number of eQTLs (365) had a consistent “static” regulatory effect on gene expression, an even larger number were found to be very sensitive to cell stage. As many as 1,283 eQTLs exhibited a “dynamic” behavior across cell types. By looking more closely at these dynamic eQTLs, we show that the sensitivity of eQTLs to cell stage is largely associated with gene expression changes in target genes. These results stress the importance of studying gene expression variation in well-defined cell populations. Only such studies will be able to reveal the important differences in gene regulation between different cell types.

FON2 SPARE1 Redundantly Regulates Floral Meristem Maintenance with FLORAL ORGAN NUMBER2 in Rice

2009-10-16T07:00:00Z

Author Summary

The body plan of plants is regulated by the function of apical meristems that are generated in the embryo. Leaves and floral organs are derived from cells supplied by stem cells in the vegetative shoot apical meristem (SAM) and the floral meristem (FM), respectively. Thus, genetic regulation of stem cell maintenance is a central issue in plant development. In the model plant Arabidopsis thaliana, CLAVATA3 (CLV3) functions as a key signaling molecule to restrict the size of the stem cell population in both the SAM and the FM. In rice, however, we show here that two CLV3-like genes, FLORAL ORGAN NUMBER2 (FON2) and FON2 SPARE1 (FOS1), redundantly regulate maintenance of the FM. We also show that FOS1 is likely to be involved in maintenance of the vegetative SAM, whereas FON2 plays no role in regulation in this meristem. FOS1 appears to act via a putative receptor that differs from the FON2 receptor, suggesting that these two signaling molecules function in independent pathways to restrict stem cells in different ways depending on the type of meristem. In addition, we show that the FOS1 gene was compromised in the standard rice, Oryza sativa spp. japonica, during the evolution of rice.