Transposase Activity Research Articles

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure.

It remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to becoming carbapenem resistant. Carbapenem mutation frequency was measured in multiple subclades of extended-spectrum β-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/ H 30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation. All thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P -value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) encoding genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing bla CTX-M-15 copy numbers via modular, insertion sequence 26 (IS 26 ) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/ H 30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

A Naturally Active Spy Transposon Discovered from the Insect Genome of Colletes gigas as a Promising Novel Gene Transfer Tool.

Novel active DNA transposons, such as Spy transposons from the PHIS superfamily, are identified through bioinformatics in this study. The native transposases cgSpy and cvSpy displayed transposition activities of approximately 85% and 35% compared to the hyperactive piggyBac transposase (hyPB). The cgSpy transposon showed unique characteristics, including a lack of overproduction inhibition and reduced efficiency for insertion sizes between 3.1 to 8.5kb. Integration preferences of cgSpy are found in genes and regulatory regions, making it suitable for genetic manipulation. Evaluation in T-cell engineering demonstrated that cgSpy-mediated chimeric antigen receptor (CAR) modification is comparable to the PB system, indicating its potential utility in cell therapy. This study unveils the promising application of the active native transposase, Spy, from Colletes gigas, as a valuable tool for genetic engineering, particularly in T-cell manipulation.

Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum

Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future.

CTCF Is a Haploinsufficient Tumor Suppressor in B-Cell Lymphoma

Alteration of epigenetic regulators is a hallmark of diffuse large B cell lymphoma (DLBCL), the most common type of non-Hodgkin B cell malignancy. It has been recently shown that haploinsufficiency of the cohesin complex ATPase subunit Smc3 cooperates with the oncogene IμBcl6 to induce lymphoma in mice, by a mechanism that involves decreased intra-topological associating domain (TAD) chromosomal interactions and downregulation of tumor suppressor genes. The transcription factor CTCF is an obligate partner of the cohesin complex in defining TADs and regulatory gene loops, acting as the boundaries of TADs and restricting the interaction of promoters and enhancers. Alterations in CTCF have been shown in several cancer types, including lymphomas. In this study, we aim to define the role of CTCF during B-cell lymphoma and lymphomagenesis. We generated germinal center (GC) B cell-specific conditional knockout mice for CTCF by crossing the CTCF floxed mice to the Ighg1-Cre (Cγ1-Cre) allele. In these experiments, we compared haploinsufficient CTCF conditional knockout mice-in which only one allele of CTCF was knocked out-versus wild-type mice expressing the Cγ1-Cre. Eight days after immunization with the T-cell-dependent antigen sheep red blood cells, we excised spleens and surveyed B cell populations by flow cytometry and immunohistochemistry. Remarkably, CTCF haploinsufficient mice contained a similar percentage of follicular GC B cells compared to wild-type mice (Live B220 +CD95 +GL7 + or live B220 +CD95 +CD38 DIM splenocytes). However, CTCF haploinsufficient GCs displayed a bias in the GC subpopulations, namely an increased percentage of light zone centrocytes (LZ: GC + markers CXCR4 loCD86 hi) and decreased dark zone centroblasts (DZ: GC + markers CXCR4 hiCD86 lo). Because biases in GCs were an early indication of malignant transformation in previous studies, we wondered whether CTCF haploinsufficiency would cooperate with lymphoma oncogenes to induce B-cell lymphoma. To address the lymphomagenic potential of CTCF haploinsufficiency, we crossed CTCF conditional knockout mice to the vavP-Bcl2 mice that develop GC-derived follicular-like lymphoma with long latency. We harvested bone marrow blood cell precursors from Cγ1-Cre (“WT”), Cγ1-Cre;CTCF +/- (“CTCF”), vavP-BCL2 +;Cγ1-Cre (“BCL2”), and vavP-BCL2 +;Cγ1-Cre;CTCF +/- (“BCL2/CTCF”) and transplanted them into lethally irradiated C57BL6/J (30 per group). Mice that survived more than two weeks after irradiation and bone marrow transplant were considered stably engrafted and monitored for any signs of disease. Lethargic mice were sacrificed and macroscopically inspected for the presence of tumors. Survival analysis by Kaplan-Meier showed that BCL2/CTCF mice had the shortest survival from all four groups, having a median survival of 230 days, while survival for the BCL2 alone group did not fall below 50% up to day 370. Immunohistochemistry against B220 + was performed in tissue sampling from dying mice to confirm the cause of death. Curiously, CTCF haploinsufficiency accelerated BCL2-induced lymphomagenesis but did not affect the survival of IμBCL6-induced lymphoma. To explore the mechanistic basis of CTCF haploinsufficiency-induced lymphoma in mice, we FACS sorted DZ and LZ GC B-cells from 8-day immunized mice and explored global transcriptomics by RNA-sequencing. While the transition from DZ to LZ involves the regulation of around ~1,500 genes, we failed to detect changes in gene expression between wild-type and CTCF haploinsufficiency cells. ATAC-seq assay on the same cells showed little to no change in chromatin accessibility between cells from CTCF haploinsufficient and wild-type mice. TOBIAS footprinting analysis, which identifies biases in Tn5 transposase activity due to protein binding, showed that CTCF haploinsufficient cells maintained a deep depletion in CTCF binding. Hi-C chromosomal conformation analysis identified an overlapping set of gene regulatory loops that lose interactions and showed decreased signal by gene set enrichment analysis. In this study, we characterized the role of CTCF haploinsufficiency phenotypically and are currently exploring the molecular mechanisms that contribute to the acceleration of the lymphomagenesis induced by the BCL2 oncogene.

IS26-mediated loss of the translocatable unit from Tn4352B requires the presence of the recA1 allele

The pseudo-compound transposon Tn4352B is unusual in that the translocatable unit (TU) consisting of one of the bounding IS26 copies and the central portion containing the aphA1a gene has been found to be readily lost in the Escherichia coli strains used as host. Rapid loss required the presence of an additional 2 G residues adjacent to the internal end of one of the IS26 that flank the central portion and an active Tnp26 transposase. However, Tn4352B was found to be stable in wild-type Klebsiella pneumoniae strains. Though it was concluded that the difference may be due to the species background, the E. coli strains used were recombination-deficient. Here, we have further investigated the requirements for TU loss in E. coli and found that Tn4352B was stable in recombination-proficient strains. Among several recombination-deficient strains examined, rapid loss occurred only in strains that carry the recA1 allele but not in strains carrying different recA alleles, recA13 and a novel recA allele identified here, that also render the strain deficient in homologous recombination. Hence, it appears that a specific property of the RecA1 protein underlies the observed TU loss from Tn4352B.

Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase.

The piggyBac DNA transposon is an active element initially isolated from the cabbage looper moth, but members of this superfamily are also present in most eukaryotic evolutionary lineages. The functionally important regions of the transposase are well described. There is an RNase H-like fold containing the DDD motif responsible for the catalytic DNA cleavage and joining reactions and a C-terminal cysteine-rich domain important for interaction with the transposon DNA. However, the protein also contains a ~100 amino acid long N-terminal disordered region (NTDR) whose function is currently unknown. Here we show that deletion of the NTDR significantly impairs piggyBac transposition, although the extent of decrease is strongly cell-type specific. Moreover, replacing the NTDR with scrambled but similarly disordered sequences did not rescue transposase activity, indicating the importance of sequence conservation. Cell-based transposon excision and integration assays reveal that the excision step is more severely affected by NTDR deletion. Finally, bioinformatic analyses indicated that the NTDR is specific for the piggyBac superfamily and is also present in domesticated, transposase-derived proteins incapable of catalyzing transposition. Our results indicate an essential role of the NTDR in the “fine-tuning” of transposition and its significance in the functions of piggyBac-originated co-opted genes.

Epigenetic Application of ATAC-Seq Based on Tn5 Transposase Purification Technology.

Background Assays of transposase accessible chromatin sequencing (ATAC-seq) is an efficient assay to investigate chromatin accessibility, which depends on the activity of a robust Tn5 transposase to fragment the genome while cutting in the sequencing adapters. Methods We propose reliable approaches for purifying hyperactive Tn5 transposase by chitin magnetic bead sorting. Double-stranded DNA of J76 cells and 293T cells were digested and subjected to tagmentation as test samples with Tn5 transposase, and libraries were established and sequenced. Sequencing data was then analyzed for peak calling, GO enrichment, and motif analysis. Results We report a set of rapid, efficient, and low-cost methods for ATAC-seq library construction and data analysis, through large-scale and rapid sequencing. These methods can provide a reference for the study of epigenetic regulation of gene expression.

Neofunctionalization of a Noncoding Portion of a DNA Transposon in the Coding Region of the Chimerical Sex-Determining Gene dm-W in Xenopus Frogs.

Most vertebrate sex-determining genes (SDGs) emerge as neofunctionalized genes through duplication and/or mutation of ancestral genes that are involved with sexual differentiation. We previously demonstrated dm-W to be the SDG in the African clawed frog Xenopus laevis and found that a portion of this gene emerged from the masculinization gene dmrt1 after allotetraploidization by interspecific hybridization between two ancestral species around 17–18 Ma. dm-W has four exons consisting of a noncoding exon 1, dmrt1-derived exons 2 and 3, and an orphan exon 4 (Ex4) of unknown origin that includes coding sequence (CDS). In this study, we searched for the origin of Ex4 and investigated the function of the CDS of this exon. We found that the Ex4-CDS is derived from a noncoding portion of the hAT-10 family of DNA transposon. Evolutionary analysis of transposons and determination of the Ex4 sequences from three other species indicated that Ex4 was generated before the diversification of most or all extant allotetraploid species in subgenus Xenopus, during which time we hypothesize that transposase activity of this hAT superfamily was active. Using DNA–protein binding and transfection assays, we further demonstrate that the Ex4-encoded amino acid sequence increases the DNA-binding ability and transrepression activity of DM-W. These findings suggest that the conversion of the noncoding transposon sequence to the CDS of dm-W contributed to neofunctionalization of a new chimeric SDG in the ancestor of the allotetraploid Xenopus species, offering new insights into de novo origin and functional evolution of chimerical genes.

Functional indications for transposase domestications – Characterization of the human piggyBac transposase derived (PGBD) activities

Transposable elements are widespread in all living organisms. In addition to self-reproduction, they are a major source of genetic variation that drives genome evolution but our knowledge of the functions of human genes derived from transposases is limited. There are examples of transposon-derived, domesticated human genes that lost (SETMAR) or retained (THAP9) their transposase activity, however, several remnants in the human genome have not been thoroughly investigated yet. These include the five human piggyBac-derived sequences (PGBD1-5) which share ancestry with the Trichoplusia ni originated piggyBac (PB) transposase. Since PB is widely used in gene delivery applications, the potential activities of endogenous PGBDs are important to address. However, previous data is controversial, especially with the claimed transposition activity of PGBD5, it awaits further investigations. Here, we aimed to systematically analyze all five human PGBD proteins from several aspects, including phylogenetic conservation, potential transposase activity, expression pattern and their regulation in different stress conditions. Among PGBDs, PGBD5 is under the highest purifying selection, and exhibits the most cell type specific expression pattern. In a two-component vector system, none of the human PGBDs could mobilize either the insect PB transposon or the endogenous human PB-like MER75 and MER85 elements with intact terminal sequences. When cells were exposed to various stress conditions, including hypoxia, oxidative or UV stress, the expression profiles of all PGBDs showed different, often cell type specific responses; however, the pattern of PGBD5 in most cases had the opposite tendency than that of the other piggyBac-derived elements. Taken together, our results indicate that human PGBD elements did not retain their mobilizing activity, but their cell type specific, and cellular stress related expression profiles point toward distinct domesticated functions that require further characterization.

AAA+ regulator MuB distorts DNA to drive forward transposition

Transposons are mobile genetic elements that drive genomic rearrangements and evolution. Many transposons, such as Tn7, CRISPR-Tn7-like, and Mu, depend on AAA+ proteins to enable target-site selection. MuB, a component of the transposition system from bacteriophage Mu, is an AAA+ protein that regulates the activity of the MuA transposase. Here, we solved a cryo-EM structure of the MuB helical filament that reveals the mechanism of its association with DNA and gives context to existing data on how MuB interacts with MuA.

Domain dynamics and plasticity of the transposon-encoded cascade-TniQ system

Cascade is a type 1, class 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. Surprisingly, the utility of these systems does not end here. Recent studies in Vibrio Cholerae have demonstrated that a transposon, Tn6677, encodes a variant of the ‘standard’ type 1F Cascade present in these systems. This variant complexes with a homodimer of the transposition protein TniQ and is responsible for directing the integration activity of a heteromeric transposase, resulting in a site-specific transposition of Tn6677.

Bacterial Adaptation by a Transposition Burst of an Invading IS Element.

The general importance of transposable elements (TEs) for adaptive evolution remains unclear. This in part reflects a poor understanding of the role of TEs for adaptation in nonmodel systems. Here, we investigated whether insertion sequence (IS) elements are a major source of beneficial mutations during 400 generations of laboratory evolution of the cyanobacterium Acaryochloris marina strain CCMEE 5410, which has experienced a recent or on-going IS element expansion and has among the highest transposase gene contents for a bacterial genome. Most mutations detected in the eight independent experimental populations were IS transposition events. Surprisingly, however, the majority of these involved the copy-and-paste activity of only a single copy of an unclassified element (ISAm1) that has recently invaded the strain CCMEE 5410 genome. ISAm1 transposition was largely responsible for the highly repeatable evolutionary dynamics observed among populations. Notably, this included mutations in multiple targets involved in the acquisition of inorganic carbon for photosynthesis that were exclusively due to ISAm1 activity. These mutations were associated with an increase in linear growth rate under conditions of reduced carbon availability but did not appear to impact fitness when carbon was readily available. Our study reveals that the activity of a single transposase can fuel adaptation for at least several hundred generations but may also potentially limit the rate of adaptation through clonal interference.

The medaka fish Tol2 transposable element is in an early stage of decay: identification of a nonautonomous copy.

The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here, we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained three local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost complete decay process that progresses in natural populations.

Genome-wide characterization of Mariner-like transposons and their derived MITEs in the Whitefly Bemisia tabaci (Hemiptera: Aleyrodidae).

The whitefly, Bemisia tabaci is a hemipteran pest of vegetable crops vectoring a broad category of viruses. Currently, this insect pest showed a high adaptability and resistance to almost all the chemical compounds commonly used for its control. In many cases, transposable elements (TEs) contributed to the evolution of host genomic plasticity. This study focuses on the annotation of Mariner-like elements (MLEs) and their derived Miniature Inverted repeat Transposable Elements (MITEs) in the genome of B. tabaci. Two full-length MLEs belonging to mauritiana and irritans subfamilies were detected and named Btmar1.1 and Btmar2.1, respectively. Additionally, 548 defective MLE sequences clustering mainly into 19 different Mariner lineages of mauritiana and irritans subfamilies were identified. Each subfamily showed a significant variation in MLE copy number and size. Furthermore, 71 MITEs were identified as MLEs derivatives that could be mobilized via the potentially active transposases encoded by Btmar 1.1 and Btmar2.1. The vast majority of sequences detected in the whitefly genome present unusual terminal inverted repeats (TIRs) of up to 400 bp in length. However, some exceptions are sequences without TIRs. This feature of the MLEs and their derived MITEs in B. tabaci genome that distinguishes them from all the other MLEs so far described in insects, which have TIRs size ranging from 20 to 40 bp. Overall, our study provides an overview of MLEs, especially those with large TIRs, and their related MITEs, as well as diversity of their families, which will provide a better understanding of the evolution and adaptation of the whitefly genome.

THAP9 Transposase Cleaves DNA via Conserved Acidic Residues in an RNaseH-Like Domain.

The catalytic domain of most ‘cut and paste’ DNA transposases have the canonical RNase-H fold, which is also shared by other polynucleotidyl transferases such as the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp—DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modeled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity has led to the identification of candidate Ds and Es in the RNaseH fold that may be a part of the catalytic triad in hTHAP9. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.

Oligomerization of THAP9 Transposase via Amino-Terminal Domains.

Active DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerization as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterized homologue of DmTNP. Here we report (using co-immunoprecipitation, pull down, colocalization, and proximity ligation assays) that both full length and truncated hTHAP9 (corresponding to amino-terminal DNA binding and predicted coiled coil domains) undergo homo-oligomerization, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerization is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerization. Thus, we highlight the importance of DNA and the amino-terminal regions of hTHAP9 for their ability to form higher-order oligomeric states. We also report that Hcf-1, THAP1, THAP10, and THAP11 are possible protein interaction partners of hTHAP9. Elucidating the functional relevance of the different putative oligomeric state(s) of hTHAP9 would help answer questions about its interaction partners as well as its unknown physiological roles.

The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases

BackgroundTransposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive.ResultsWe provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state.ConclusionsThe present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

ATRX limits the accessibility of histone H3-occupied HSV genomes during lytic infection.

Histones are rapidly loaded on the HSV genome upon entry into the nucleus of human fibroblasts, but the effects of histone loading on viral replication have not been fully defined. We showed recently that ATRX is dispensable for de novo deposition of H3 to HSV genomes after nuclear entry but restricted infection through maintenance of viral heterochromatin. To further investigate the roles that ATRX and other histone H3 chaperones play in restriction of HSV, we infected human fibroblasts that were systematically depleted of nuclear H3 chaperones. We found that the ATRX/DAXX complex is unique among nuclear H3 chaperones in its capacity to restrict ICP0-null HSV infection. Only depletion of ATRX significantly alleviated restriction of viral replication. Interestingly, no individual nuclear H3 chaperone was required for deposition of H3 onto input viral genomes, suggesting that during lytic infection, H3 deposition may occur through multiple pathways. ChIP-seq for total histone H3 in control and ATRX-KO cells infected with ICP0-null HSV showed that HSV DNA is loaded with high levels of histones across the entire viral genome. Despite high levels of H3, ATAC-seq analysis revealed that HSV DNA is highly accessible, especially in regions of high GC content, and is not organized largely into ordered nucleosomes during lytic infection. ATRX reduced accessibility of viral DNA to the activity of a TN5 transposase and enhanced accumulation of viral DNA fragment sizes associated with nucleosome-like structures. Together, these findings support a model in which ATRX restricts viral infection by altering the structure of histone H3-loaded viral chromatin that reduces viral DNA accessibility for transcription. High GC rich regions of the HSV genome, especially the S component inverted repeats of the HSV-1 genome, show increased accessibility, which may lead to increased ability to transcribe the IE genes encoded in these regions during initiation of infection.

The C-terminal Domain of piggyBac Transposase Is Not Required for DNA Transposition

PiggyBac(PB)-like elements (pble) are members of a eukaryotic DNA transposon family. This family is of interest to evolutionary genomics because pble transposases have been domesticated at least 9 times in vertebrates. The amino acid sequence of pble transposases can be split into three regions: an acidic N-terminal domain (~100 aa), a central domain (~400 aa) containing a DD[D/E] catalytic triad, and a cysteine-rich domain (CRD; ~90 aa). Two recent reports suggested that a functional CRD is required for pble transposase activity. Here we found that two CRD-deficient pble transposases, a PB variant and an isoform encoded by the domesticated PB-derived vertebrate transposase gene 5 (pgbd5) trigger transposition of the Ifp2 pble. When overexpressed in HeLa cells, these CRD-deficient transposases can insert Ifp2 elements with proper and improper transposon ends, associated with deleterious effects on cells. Finally, we found that mouse CRD-deficient transposase Pgbd5, as well as PB, do not insert pbles at random into chromosomes. Transposition events occurred more often in genic regions, in the neighbourhood of the transcription start sites and were often found in genes predominantly expressed in the human central nervous system.

Adaptation induced by self-targeting in a type I-B CRISPR-Cas system

Haloferax volcanii is, to our knowledge, the only prokaryote known to tolerate CRISPR-Cas–mediated damage to its genome in the WT background; the resulting cleavage of the genome is repaired by homologous recombination restoring the WT version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalyzed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centered on the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in WT Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2, and Cas4. Taken together, these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.