Transposon Elements Research Articles

Epigenetic Regulation Shapes the Stem Cells State.

Epigenetic Regulation Shapes the Stem Cells State.

Detecting dispersed duplications in high-throughput sequencing data using a database-free approach.

Dispersed duplications (DDs) such as transposon element insertions and copy number variations are ubiquitous in the human genome. They have attracted the interest of biologists as well as medical researchers due to their role in both evolution and disease. The efforts of discovering DDs in high-throughput sequencing data are currently dominated by database-oriented approaches that require pre-existing knowledge of the DD elements to be detected. We present DD_DETECTION, a database-free approach to finding DD events in high-throughput sequencing data. DD_DETECTION is able to detect DDs purely from paired-end read alignments. We show in a comparative study that this method is able to compete with database-oriented approaches in recovering validated transposon insertion events. We also experimentally validate the predictions of DD_DETECTION on a human DNA sample, showing that it can find not only duplicated elements present in common databases but also DDs of novel type. The software presented in this article is open source and available from https://bitbucket.org/mkroon/dd_detection.

Putative adaptive inter-slope divergence of transposon frequency in fruit flies (Drosophila melanogaster) at "Evolution Canyon", Mount Carmel, Israel.

BackgroundThe current analysis of transposon elements (TE) in Drosophila melanogaster at Evolution Canyon, (EC), Israel, is based on data and analysis done by our collaborators (Drs. J. Gonzalez, J. Martinez and W. Makalowski, this issue). They estimated the frequencies of 28 TEs (transposon elements) in fruit flies (D. melanogaster) from the ecologically tropic, hot, and dry south-facing slope (SFS) or “African” slope (AS) of EC and compared it with the TE frequencies on the temperate-cool and humid north-facing slope (NFS) or “European” slope (ES), separated, on average, by 250 m. The flies were sampled from two stations on each slope. We received their results, including the frequencies of each TE on each slope, and the probabilities of the statistical analyses (G-tests) of each TE separately. We continued the analysis of the inter-slope differences of the frequencies of the TEs, and based our different conclusions on that analysis and on the difference between micro (=EC) and macro (2000 km.) comparisons [Gonzalez et al. 2015 doi:10.1186/s13062-015-0075-4].ResultsOur collaborators based all their conclusions on the non-significant results of each of the individual tests of the 28 TEs. We analysed also the distribution of the TE differences between the slopes, based on their results. Thirteen TEs were more frequent on the SFS, 11 were more frequent on the NFS, and four had equal frequencies. Because of the equalizing effect of the ongoing migration, only small and temporary differences between the slopes (0 – 0.06) were regarded by us as random fluctuations (drift). Three TEs were intermediate (0.08-0.09) and await additional research. The 11 TEs with large frequency differences (0.12 – 0.22) were regarded by us as putative adaptive TEs, because the equalizing power of ongoing migration will eliminate random large differences. Five of them were higher on the SFS and six were higher on the NFS. Gaps in the distribution of the differences distinguished between the large and small differences. The large gap among the 11 TEs favored on the NFS was significant and supports our rejection of drift as the only explanation of the distribution of the slope differences. The gaps in the distribution of the differences separated the putative TEs with strong enough selection from those TEs that couldn't overrule the migration.The results are compared and contrasted with the directional effect of the frequencies of the same TEs in the study of global climatic comparisons across thousands of kilometers. From the 11 putative adaptive TEs in the local “Evolution Canyon,” six differentiate in the same direction as in the continental comparisons and four in the opposite direction. One TE, FBti0019144, differentiated in EC in the same direction as in Australia and in the opposite direction to that of North America.ConclusionsWe presume that the major divergent evolutionary driving force at the local EC microsite is natural selection overruling gene flow. Therefore, after we rejected drift as an explanation of all the large slope differences, we regarded them as putatively adaptive. In order to substantiate the individual TE adaptation, we need to increase the sample sizes and reveal the significant adaptive TEs.The comparison of local and global studies show only partial similarity in the adaptation of the TEs, because of the dryness of the ecologically tropical climate in EC, in contrast to the wet tropical climate in the global compared climates. Moreover, adaptation of a TE may be expressed only in part of the time and specific localities.ReviewersReviewed by Eugene Koonin, Limsoon Wong and Fyodor Kondrashov. For the full reviews, please go to the Reviewers’ comments section.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0074-5) contains supplementary material, which is available to authorized users.

Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice.

Heritable epigenetic variants of genes, termed epialleles, can broaden genetic and phenotypic diversity in eukaryotes. Epialleles may also provide a new source of beneficial traits for crop breeding, but very few epialleles related to agricultural traits have been identified in crops. Here, we identified Epi-rav6, a gain-of-function epiallele of rice (Oryza sativa) RELATED TO ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1) 6 (RAV6), which encodes a B3 DNA-binding domain-containing protein. The Epi-rav6 plants show larger lamina inclination and smaller grain size; these agronomically important phenotypes are inherited in a semidominant manner. We did not find nucleotide sequence variation of RAV6. Instead, we found hypomethylation in the promoter region of RAV6, which caused ectopic expression of RAV6 in Epi-rav6 plants. Bisulfite analysis revealed that cytosine methylation of four CG and two CNG loci within a continuous 96-bp region plays essential roles in regulating RAV6 expression; this region contains a conserved miniature inverted repeat transposable element transposon insertion in cultivated rice genomes. Overexpression of RAV6 in the wild type phenocopied the Epi-rav6 phenotype. The brassinosteroid (BR) receptor BR INSENSITIVE1 and BR biosynthetic genes EBISU DWARF, DWARF11, and BR-DEFICIENT DWARF1 were ectopically expressed in Epi-rav6 plants. Also, treatment with a BR biosynthesis inhibitor restored the leaf angle defects of Epi-rav6 plants. This indicates that RAV6 affects rice leaf angle by modulating BR homeostasis and demonstrates an essential regulatory role of epigenetic modification on a key gene controlling important agricultural traits. Thus, our work identifies a unique rice epiallele, which may represent a common phenomenon in complex crop genomes.

Abstract 4192: Identification of novel genetic drivers of vulvar carcinoma in a Sleeping Beauty model of spontaneous cancer

Abstract Introduction: The purpose of this study was to develop a Sleeping Beauty (SB)-induced mouse model of metastatic vulvar carcinoma with which we could identify novel tumor-driving mutations. Experimental Procedures: An existing transgenic mouse model known to induce development of benign vaginal papillomas, (PrCre/+;KrasG12D), was bred to mice containing the components of the SB system to render (PrCre/+;KrasG12D;SB) mice. The SB system consists of a transposable element (transposon) and an activating enzyme (transposase) that induces excision of the transposon from one site to another in the host cell genome. Insertional mutations that enhance cell survival and proliferation are selected for, resulting in the development of cancer. We utilize the SB system to identify candidate driver mutations since the transposon is not only the mutagen but also serves as a sequence tag for localization of mutations. Mice were aged and monitored for phenotypic signs of disease. Upon necropsy, gynecologic and other grossly abnormal organs were harvested. Genomic DNA was extracted from each sample, prepared for genetic analysis, and sequenced using the Illumina platform. Candidate genes were identified using an unbiased Monte-Carlo approach in conjunction with a gene-centric Chi-Squared analysis. Paraffin-embedded human tumor samples were obtained from the University of Iowa Hospitals and Clinics for immunohistochemical labeling of protein expression for selected genes. Results: Vulvar carcinoma developed in 29 of 29 PrCre/+;KrasG12D;SB female mice with metastases by seven months of age confirmed in 13 animals. Organs within the abdominal cavity including the uterus and ovaries contained metastatic lesions in some mice. In one mouse, a distant metastasis within the subcutaneous tissue of the thoracic back was observed. Illumina sequencing of primary lesions from all mice revealed 77 candidate tumor drivers. Conclusions: To our knowledge, the PrCre/+;KrasG12D;SB mouse is the first reported metastatic model of vulvar carcinoma. Protein expression of candidate genes identified by our SB screen are being assessed in human tissue samples. Patient clinical data will be incorporated into analyses to determine whether candidate genes are correlated to patient outcomes. This work will permit insights into the pathophysiology and future treatment of vulvar cancer. Note: This abstract was not presented at the meeting. Citation Format: Amy Guimaraes-Young, Traci Neff, David K. Meyerholz, Adam J. Dupuy, Michael J. Goodheart. Identification of novel genetic drivers of vulvar carcinoma in a Sleeping Beauty model of spontaneous cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4192. doi:10.1158/1538-7445.AM2015-4192

Epigenetics and Evolution: Transposons and the Stochastic Epigenetic Modification Model

AbstractIn addition to genetic variation, epigenetic variation and transposons can greatly affect the evolutionary fitnesses landscape and gene expression. Previously we proposed a mathematical treatment of a general epigenetic variation model that we called Stochastic Epigenetic Modification (SEM) model. In this study we follow up with a special case, the Transposon Silencing Model (TSM), with, once again, emphasis on quantitative treatment. We have investigated the evolutionary effects of epigenetic changes due to transposon (T) insertions; in particular, we have considered a typical gene locus A and postulated that (i) the expression level of gene A depends on the epigenetic state (active or inactive) of a cis-located transposon element T, (ii) stochastic variability in the epigenetic silencing of T occurs only in a short window of opportunity during development, (iii) the epigenetic state is then stable during further development, and (iv) the epigenetic memory is fully reset at each generation. We develop the model using two complementary approaches: a standard analytical population genetics framework (diffusion equations) and Monte-Carlo simulations. Both approaches led to similar estimates for the probability of fixation and time of fixation of locus TA with initial frequency P in a randomly mating diploid population of effective size Ne . We have ascertained the effect that ρ, the probability of transposon modification during the developmental window, has on the population (species). One of our principal conclusions is that as ρ increases, the pattern of fixation of the combined TA locus goes from “neutral” to “dominant” to “over-dominant”. We observe that, under realistic values of ρ, epigenetic modifications can provide an efficient mechanism for more rapid fixation of transposons and cis-located gene alleles. The results obtained suggest that epigenetic silencing, even if strictly transient (being reset at each generation), can still have significant macro-evolutionary effects. Importantly, this conclusion also holds for the static fitness landscape. To the best of our knowledge, no previous analytical modeling has treated stochastic epigenetic changes during a window of opportunity.

Chromatin structure analysis enables detection of DNA insertions into the mammalian nuclear genome

BackgroundGenetically modified organisms (GMOs) have numerous biomedical, agricultural and environmental applications. Development of accurate methods for the detection of GMOs is a prerequisite for the identification and control of authorized and unauthorized release of these engineered organisms into the environment and into the food chain. Current detection methods are unable to detect uncharacterized GMOs, since either the DNA sequence of the transgene or the amino acid sequence of the protein must be known for DNA-based or immunological-based detection, respectively. MethodsHere we describe the application of an epigenetics-based approach for the detection of mammalian GMOs via analysis of chromatin structural changes occurring in the host nucleus upon the insertion of foreign or endogenous DNA. ResultsImmunological methods combined with DNA next generation sequencing enabled direct interrogation of chromatin structure and identification of insertions of various size foreign (human or viral) DNA sequences, DNA sequences often used as genome modification tools (e.g. viral sequences, transposon elements), or endogenous DNA sequences into the nuclear genome of a model animal organism. ConclusionsThe results provide a proof-of-concept that epigenetic approaches can be used to detect the insertion of endogenous and exogenous sequences into the genome of higher organisms where the method of genetic modification, the sequence of inserted DNA, and the exact genomic insertion site(s) are unknown. General significanceMeasurement of chromatin dynamics as a sensor for detection of genomic manipulation and, more broadly, organism exposure to environmental or other factors affecting the epigenomic landscape are discussed.

P Transposable Elements in Drosophila and other Eukaryotic Organisms.

P transposable elements were discovered in Drosophila as the causative agents of a syndrome of genetic traits called hybrid dysgenesis. Hybrid dysgenesis exhibits a unique pattern of maternal inheritance linked to the germline-specific small RNA piwi-interacting (piRNA) pathway. The use of P transposable elements as vectors for gene transfer and as genetic tools revolutionized the field of Drosophila molecular genetics. P element transposons have served as a useful model to investigate mechanisms of cut-and-paste transposition in eukaryotes. Biochemical studies have revealed new and unexpected insights into how eukaryotic DNA-based transposons are mobilized. For example, the P element transposase makes unusual 17nt-3' extended double-strand DNA breaks at the transposon termini and uses guanosine triphosphate (GTP) as a cofactor to promote synapsis of the two transposon ends early in the transposition pathway. The N-terminal DNA binding domain of the P element transposase, called a THAP domain, contains a C2CH zinc-coordinating motif and is the founding member of a large family of animal-specific site-specific DNA binding proteins. Over the past decade genome sequencing efforts have revealed the presence of P element-like transposable elements or P element transposase-like genes (called THAP9) in many eukaryotic genomes, including vertebrates, such as primates including humans, zebrafish and Xenopus, as well as the human parasite Trichomonas vaginalis, the sea squirt Ciona, sea urchin and hydra. Surprisingly, the human and zebrafish P element transposase-related THAP9 genes promote transposition of the Drosophila P element transposon DNA in human and Drosophila cells, indicating that the THAP9 genes encode active P element "transposase" proteins.

English

Drought is one of the main constraints facing crops and affecting their production. In a trial to investigate and understand drought effect, differential display experiments were performed where; about 107 DD-fragments have been isolated from wild barley plants subjected to drought treatment. These fragments were categorized according to their gene expression into four categories: 1) up regulated genes; 2) up and down regulated; 3) down regulated and 4) down and up regulated genes. The isolated fragments were cloned and sequenced. Sequence analysis using GeneBank database revealed that some fragments have significant similarities with starch branching enzyme I (sbeI) gene; glycosyl transferase family 1 gene; Retrotransposons Ty3-gypsy subclass; gag-polypeptide of LTR copia-type retroelement gene; IMP cyclohydrolase/phosphoribosylaminoimidazolecarboxamide formyltransferase and unknown proteins. Some other fragments have significant similarity with Triticum aestivum gene for TaAP2-B, complete cds and 3B chromosome, clone BAC TA3B63N2. These results indicate that starch metabolism, transposon elements and purine metabolism might have been affected by drought stress. Key words: Wild barley, differential display, drought; starch metabolism, purine metabolism.

Regulatory link between DNA methylation and active demethylation in Arabidopsis

De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5' UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression.

Construction of Adoxophyes honmai nucleopolyhedrovirus bacmid DNA by using a gene-repair BAC cassette

Recombinant baculoviruses have traditionally been generated by homologous recombination, in insect cells, of the virus DNA and a transfer vector. This method is, however, restricted to the small number of baculoviruses for which permissive cell lines are available. An alternative method, which avoids this limitation, is the bacmid system, in which a bacterial DNA cassette (BAC cassette) comprising a bacterial replication origin, an antibiotic resistance marker gene, and a transposon element, is inserted into a unique restriction endonuclease site of the virus genome and the recombinant DNA is produced in Escherichia coli K-12 strain. However, if the unique cleavage site is located within an open reading frame (ORF), the ORF will be disrupted by BAC cassette insertion. In this study, we propose a gene-repair BAC method which simultaneously restores the disrupted ORF and inserts a BAC cassette into the full genome of Adoxophyes honmai nucleopolyhedrovirus.

Interactions of Chromatin Context, Binding Site Sequence Content, and Sequence Evolution in Stress-Induced p53 Occupancy and Transactivation

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10−7) and correlated with stronger p53RE sequences (p<10−110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.

Assessment of RNAi-induced silencing in banana (Musa spp.).

BackgroundIn plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana.ResultsUsing embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6–8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target sequences (26-nt and 19-nt).ConclusionsRNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-655) contains supplementary material, which is available to authorized users.

A piggyBac transposon‐ and gateway‐enhanced system for efficient BAC transgenesis

Background: Bacterial artificial chromosomes (BACs) have become increasingly popular vectors for making transgenic mice, as they are able to carry large genomic DNA fragments that in many cases are needed to reproduce the endogenous gene expression pattern. However, the efficiency of BAC transgenesis is generally low, and gene transfer to BAC vectors by recombination‐mediated engineering (recombineering) is time‐consuming and technically demanding. Results and Conclusions: We present an enhanced system, comprising a BAC vector retrofitted with piggyBac DNA transposon elements and attL (Gateway) docking sites, that obviates these problems. Using this system, a gene‐of‐interest (such as a reporter gene) is transferred to the vector in a one‐step in vitro reaction, and piggyBac transposition mediates transgene integration at high efficiency when microinjected into mouse zygotes with piggyBac transposase mRNA. We establish proof‐of‐principle for this system using a Wilms tumour‐1 (Wt1) BAC to drive expression of an mCherry‐2A‐EGFP (RG) reporter gene, which yielded transgenic mice at a frequency of 33%, and recapitulated endogenous WT1 expression in developing gonads, kidneys and heart. The system we describe is applicable to any BAC transgenesis strategy. Developmental Dynamics 243:1086–1094, 2014. © 2014 Wiley Periodicals, Inc.

Structural studies of Rhino protein in piRNA biogenesis

Small-RNA-guided gene regulation is a common biological process in eukaryotic cells. Animal germ cells are characterized by an intriguing small-RNA-mediated gene-silencing mechanism known as PIWI pathway. PIWI-interacting RNAs (piRNAs) are small, 21-30 nt single-stranded RNAs that associate with PIWI proteins. The function of piRNA is silencing transposon elements in germ line cells to keep the genome integrity since germ line cells are the only source for transmitting genetic information to the next generation. For a long time the biogenesis of piRNA and the mechanism of how it functions remains unclear. The biogenesis of piRNAs is quite different from that of other small-RNA pathways, which is independent of Dicer. piRNA biogenesis occurs through both primary and secondary pathway (or called ping-pong cycle). In drosophila transcripts from heterochromatic clusters are processed into primary piRNAs. A particularly fast evolving homologue of heterochromatin protein 1 (HP1) called Rhino binds to dual-strand piRNA clusters and is required for their production. But how does Rhino recognize histone H3 trimethylated on lysine 9? What's the difference between Rhino and other HP1 proteins? Here we show the crystal structure of Rhino both in apo form and complex form with H3K9me3. We observed a unique dimer interface in Rhino and a domain-swapping in conformational change. These findings provide insights into the molecular mechanism of the specificity of Rhino recognizing histone H3K9me3 and its function in piRNA biogenesis.

Non-exhaustive DNA methylation-mediated transposon silencing in the black truffle genome, a complex fungal genome with massive repeat element content.

BackgroundWe investigated how an extremely transposon element (TE)-rich organism such as the plant-symbiotic ascomycete truffle Tuber melanosporum exploits DNA methylation to cope with the more than 45,000 repeated elements that populate its genome.ResultsWhole-genome bisulfite sequencing performed on different developmental stages reveals a high fraction of methylated cytosines with a strong preference for CpG sites. The methylation pattern is highly similar among samples and selectively targets TEs rather than genes. A marked trend toward hypomethylation is observed for TEs located within a 1 kb distance from expressed genes, rather than segregated in TE-rich regions of the genome. Approximately 300 hypomethylated or unmethylated TEs are transcriptionally active, with higher expression levels in free-living mycelium compared to fruitbody. Indeed, multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs are found almost exclusively in free-living mycelium. A reduction of DNA methylation, restricted to non-CpG sites and accompanied by an increase in TE expression, is observed upon treatment of free-living mycelia with 5-azacytidine.ConclusionsEvidence derived from analysis of the T. melanosporum methylome indicates that a non-exhaustive, partly reversible, methylation process operates in truffles. This allows for the existence of hypomethylated, transcriptionally active TEs that are associated with copy number variant regions of the genome. Non-exhaustive TE methylation may reflect a role of active TEs in promoting genome plasticity and the ability to adapt to sudden environmental changes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0411-5) contains supplementary material, which is available to authorized users.

A piggyBac transposon- and gateway-enhanced system for efficient BAC transgenesis.

Bacterial artificial chromosomes (BACs) have become increasingly popular vectors for making transgenic mice, as they are able to carry large genomic DNA fragments that in many cases are needed to reproduce the endogenous gene expression pattern. However, the efficiency of BAC transgenesis is generally low, and gene transfer to BAC vectors by recombination-mediated engineering (recombineering) is time-consuming and technically demanding. We present an enhanced system, comprising a BAC vector retrofitted with piggyBac DNA transposon elements and attL (Gateway) docking sites, that obviates these problems. Using this system, a gene-of-interest (such as a reporter gene) is transferred to the vector in a one-step in vitro reaction, and piggyBac transposition mediates transgene integration at high efficiency when microinjected into mouse zygotes with piggyBac transposase mRNA. We establish proof-of-principle for this system using a Wilms tumour-1 (Wt1) BAC to drive expression of an mCherry-2A-EGFP (RG) reporter gene, which yielded transgenic mice at a frequency of 33%, and recapitulated endogenous WT1 expression in developing gonads, kidneys and heart. The system we describe is applicable to any BAC transgenesis strategy.

Transposon Mutagenesis of Probiotic Lactobacillus casei Identifies asnH, an Asparagine Synthetase Gene Involved in Its Immune-Activating Capacity

Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

Non-exhaustive DNA methylation-mediated transposon silencing in the black truffle genome, a complex fungal genome with massive repeat element content

BackgroundWe investigated how an extremely transposon element (TE)-rich organism such as the plant-symbiotic ascomycete truffle Tuber melanosporum exploits DNA methylation to cope with the more than 45,000 repeated elements that populate its genome.ResultsWhole-genome bisulfite sequencing performed on different developmental stages reveals a high fraction of methylated cytosines with a strong preference for CpG sites. The methylation pattern is highly similar among samples and selectively targets TEs rather than genes. A marked trend toward hypomethylation is observed for TEs located within a 1 kb distance from expressed genes, rather than segregated in TE-rich regions of the genome. Approximately 300 hypomethylated or unmethylated TEs are transcriptionally active, with higher expression levels in free-living mycelium compared to fruitbody. Indeed, multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs are found almost exclusively in free-living mycelium. A reduction of DNA methylation, restricted to non-CpG sites and accompanied by an increase in TE expression, is observed upon treatment of free-living mycelia with 5-azacytidine.ConclusionsEvidence derived from analysis of the T. melanosporum methylome indicates that a non-exhaustive, partly reversible, methylation process operates in truffles. This allows for the existence of hypomethylated, transcriptionally active TEs that are associated with copy number variant regions of the genome. Non-exhaustive TE methylation may reflect a role of active TEs in promoting genome plasticity and the ability to adapt to sudden environmental changes.

Distribution of Conventional and Nonconventional Introns in Tubulin (α and β) Genes of Euglenids

The nuclear genomes of euglenids contain three types of introns: conventional spliceosomal introns, nonconventional introns for which a splicing mechanism is unknown (variable noncanonical borders, RNA secondary structure bringing together intron ends), and so-called intermediate introns, which combine features of conventional and nonconventional introns. Analysis of two genes, tubA and tubB, from 20 species of euglenids reveals contrasting distribution patterns of conventional and nonconventional introns—positions of conventional introns are conserved, whereas those of the nonconventional ones are unique to individual species or small groups of closely related taxa. Moreover, in the group of phototrophic euglenids, 11 events of conventional intron loss versus 15 events of nonconventional intron gain were identified. A comparison of all nonconventional intron sequences highlighted the most conserved elements in their sequence and secondary structure. Our results led us to put forward two hypotheses. 1) The first one posits that mutational changes in intron sequence could lead to a change in their excision mechanism—intermediate introns would then be a transitional form between the conventional and nonconventional introns. 2) The second hypothesis concerns the origin of nonconventional introns—because of the presence of inverted repeats near their ends, insertion of MITE-like transposon elements is proposed as a possible source of new introns.