DDE Domain Research Articles

Investigation of Pogz Gene Variants in Non-Syndromic Autism Spectrum Disorder.

Genetic factors play an important role in the etiopathogenesis of autism spectrum disorder (ASD). The Pogo Transposable Element with ZNF Domain protein (POGZ) gene (MIM*614787) has been reported to be one of the most frequently mutated genes associated with ASD. This study aims to analyze the exonic regions of the POGZ gene in individuals diagnosed with non-syndromic ASD. Fifty-one non-syndromic cases diagnosed with ASD according to the DSM-V diagnostic criteria, aged 2-18 years, were included in the study. The healthy control group consisted of 50 children of similar age groups without neurodevelopmental problems. Amplicons produced using deep intronic primers covering the mRNA-encoded regions of the POGZ gene from at least 50 base pairs were sequenced by Next Generation Sequencing Analysis. No pathogenic or likely pathogenic variants reported in open-access databases (ClinVar, HGMD, etc.) were detected in the case group. In the ASD and healthy control groups, rs113396244, rs11204811, rs779479223, rs772352054, rs3831142, rs112072925, rs227453 and rs142860188 variants were determined. The rs3831142, rs112072925, rs2274535, rs142860188 variants were found statistically significant in the ASD group. The distribution of the cases with detected single nucleotide polymorphisms (SNPs) according to gender was not statistically significant. The variants identified as statistically significant within the patient group are situated in regions that encompass both the HP1-ZNF and DDE domains of the protein. Given the crucial role that the DDE domain plays, particularly in fetal brain development and neurogenesis, these four variants may potentially possess modifying and/or predisposing effects in the context of ASD.

Hiker, a new family of DNA transposons encoding transposases with DD35E motifs, displays a distinct phylogenetic relationship with most known DNA transposon families of IS630-Tc1-mariner (ITm)

DNA transposons play a crucial role in determining the size and structure of eukaryotic genomes. In this study, a new family of IS630-Tc1-mariner (ITm) DNA transposons, named Hiker (HK), was identified. HK is characterized by a DD35E catalytic domain and is distinct from all previously known families of the ITm group. Phylogenetic analyses showed that DD35E/Hiker forms a monophyletic clade with DD34E/Gambol, indicating that they may represent a separate superfamily of ITm. A total of 178 Hiker species were identified, with 170 found mainly in Actinopterygii, one in Chondrichthyes, six in Anura and one in Mollusca. Gambol (GM), on the other hand, are found in invertebrates, with 18 in Arthropoda and one in Platyhelminthes. Hiker transposons have a total length ranging from 2.14 to 3.67 kb and contain a single open reading frame that encodes a protein of approximately 370 amino acids (range 311–413 aa). They are flanked by short terminal inverted repeats (TIRs) of 16–30 base pairs and two base pair (TA) target-site duplications. In contrast, most transposons of the Gambol family have a total length of 1.35–5.96 kb, encode a transposase protein of approximately 350 amino acids (range 306–374 aa), and are flanked by TIRs that range from 32 to 1097 bp in length. Both Hiker and Gambol transposases have several conserved motifs, including helix-turn-helix (HTH) motifs and a DDE domain. Our study observed multiple amplification waves and repeated horizontal transfer (HT) events of HK transposons in vertebrate genomes, indicating their role in diversifying and shaping the genomes of Actinopterygii, Chondrichthyes, and Anura. Conversely, GM transposons showed few Horizontal transfer events. According to cell-based transposition assays, most HK transposons are likely inactive due to the truncated DNA binding domains of their transposases. We present an updated classification of the ITm group based on these findings, which will enhance the understanding of both the evolution of ITm transposons and that of their hosts.

Insertion sequences related to ISAjo2 target pdif and dif sites and belong to a new IS family, the IS1202 family.

Several insertion sequences (IS) found in various Acinetobacter species exhibit target specificity. They are found, in the same orientation, 5 bp from the XerC binding site of the pdif sites associated with dif modules in Acinetobacter plasmids, and searches revealed they are also found near chromosomal dif sites of Acinetobacter species. These IS are 1.5 kb long, bounded by 24-26 bp imperfect terminal inverted repeats (TIRs) and encode a large transposase of 441-457 aa. They generate 5 bp target site duplications (TSDs). Structural predictions of the ISAjo2 transposase, TnpAjo2, modelled on TnsB of Tn7 revealed two N-terminal HTH domains followed by an RNaseH fold (DDE domain), a β barrel and a C-terminal domain. Similar to Tn7, the outer IS ends are 5'-TGT and ACA-3', and an additional Tnp binding site, corresponding to the internal portion of the IR, is found near each end. However, the Acinetobacter IS do not encode further proteins related to those required by Tn7 for targeted transposition, and the transposase may interact directly with XerC bound to a dif-like site. We propose that these IS, currently in the IS1202 group in the not characterized yet (NCY) category in ISFinder, are part of a distinct IS1202 family. Other IS listed as in the IS1202 group encode transposases related to TnpAjo2 (25-56 % amino acid identity) and have similar TIRs but fall into three groups based on the TSD length (3-5, >15, 0 bp). Those with 3-5 bp TSDs may also target dif-like sites but targets were not found for the other groups.

Traveler, a New DD35E Family of Tc1/Mariner Transposons, Invaded Vertebrates Very Recently.

The discovery of new members of the Tc1/mariner superfamily of transposons is expected based on the increasing availability of genome sequencing data. Here, we identified a new DD35E family termed Traveler (TR). Phylogenetic analyses of its DDE domain and full-length transposase showed that, although TR formed a monophyletic clade, it exhibited the highest sequence identity and closest phylogenetic relationship with DD34E/Tc1. This family displayed a very restricted taxonomic distribution in the animal kingdom and was only detected in ray-finned fish, anura, and squamata, including 91 vertebrate species. The structural organization of TRs was highly conserved across different classes of animals. Most intact TR transposons had a length of ∼1.5 kb (range 1,072–2,191 bp) and harbored a single open reading frame encoding a transposase of ∼340 aa (range 304–350 aa) flanked by two short-terminal inverted repeats (13–68 bp). Several conserved motifs, including two helix-turn-helix motifs, a GRPR motif, a nuclear localization sequence, and a DDE domain, were also identified in TR transposases. This study also demonstrated the presence of horizontal transfer events of TRs in vertebrates, whereas the average sequence identities and the evolutionary dynamics of TR elements across species and clusters strongly indicated that the TR family invaded the vertebrate lineage very recently and that some of these elements may be currently active, combining the intact TR copies in multiple lineages of vertebrates. These data will contribute to the understanding of the evolutionary history of Tc1/mariner transposons and that of their hosts.

A Mos1 transposase in vivo assay to screen new HIV-1 integrase inhibitors.

The integrase and transposase enzymes of retrovirus and transposons, respectively, share the catalytic DDE domain. In vitro assays showed that inhibitors of HIV-1 integrase generally inhibit the mariner Mos1 transposase. Using a Drosophila strain in which the mobilisation of the mariner element can be quantified by mosaic eyes, we showed that flies maintained in medium containing 210µM to 4mM of raltegravir, or 1 or 2mM of dolutegravir, which are HIV-1 integrase inhibitor used in AIDS treatment, have 23-33% less somatic mobilisation in mosaic eyes when treated with raltegravir and 28-32% when treated with dolutegravir. The gene expression of the mariner transposase gene, estimated by qPCR, is similar among treated and control flies. The results suggest that in vivo assays using Drosophila can be used as a primary screening of inhibitory drugs for transposase and retroviral integrase. The advantages of this assay are that it is easy, quick, cheap and is an in vivo test, meaning that the tested substance has to have been taken in by cells and has arrived at the target site, which is not the case when in vitro assays are applied.

Structural Determinants of Sleeping Beauty Transposase Activity

Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.

Ginger DNA transposons in eukaryotes and their evolutionary relationships with long terminal repeat retrotransposons

BackgroundIn eukaryotes, long terminal repeat (LTR) retrotransposons such as Copia, BEL and Gypsy integrate their DNA copies into the host genome using a particular type of DDE transposase called integrase (INT). The Gypsy INT-like transposase is also conserved in the Polinton/Maverick self-synthesizing DNA transposons and in the 'cut and paste' DNA transposons known as TDD-4 and TDD-5. Moreover, it is known that INT is similar to bacterial transposases that belong to the IS3, IS481, IS30 and IS630 families. It has been suggested that LTR retrotransposons evolved from a non-LTR retrotransposon fused with a DNA transposon in early eukaryotes. In this paper we analyze a diverse superfamily of eukaryotic cut and paste DNA transposons coding for INT-like transposase and discuss their evolutionary relationship to LTR retrotransposons.ResultsA new diverse eukaryotic superfamily of DNA transposons, named Ginger (for 'Gypsy INteGrasE Related') DNA transposons is defined and analyzed. Analogously to the IS3 and IS481 bacterial transposons, the Ginger termini resemble those of the Gypsy LTR retrotransposons. Currently, Ginger transposons can be divided into two distinct groups named Ginger1 and Ginger2/Tdd. Elements from the Ginger1 group are characterized by approximately 40 to 270 base pair (bp) terminal inverted repeats (TIRs), and are flanked by CCGG-specific or CCGT-specific target site duplication (TSD) sequences. The Ginger1-encoded transposases contain an approximate 400 amino acid N-terminal portion sharing high amino acid identity to the entire Gypsy-encoded integrases, including the YPYY motif, zinc finger, DDE domain, and, importantly, the GPY/F motif, a hallmark of Gypsy and endogenous retrovirus (ERV) integrases. Ginger1 transposases also contain additional C-terminal domains: ovarian tumor (OTU)-like protease domain or Ulp1 protease domain. In vertebrate genomes, at least two host genes, which were previously thought to be derived from the Gypsy integrases, apparently have evolved from the Ginger1 transposase genes. We also introduce a second Ginger group, designated Ginger2/Tdd, which includes the previously reported DNA transposon TDD-4.ConclusionsThe Ginger superfamily represents eukaryotic DNA transposons closely related to LTR retrotransposons. Ginger elements provide new insights into the evolution of transposable elements and certain transposable element (TE)-derived genes.

Lens Epithelium-derived Growth Factor/p75 Interacts with the Transposase-derived DDE Domain of PogZ

Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular interaction partner of human immunodeficiency virus-1 (HIV-1) integrase, tethering the preintegration complex to the host chromosome. In light of the development of LEDGF/p75-integrase interaction inhibitors, it is essential to understand the cell biology of LEDGF/p75. We identified pogZ as new cellular interaction partner of LEDGF/p75. Analogous to lentiviral integrase, pogZ, a domesticated transposase, carries a DDE domain, the major determinant for LEDGF/p75 interaction. Using different in vitro and in vivo approaches, we corroborated the interaction between the C terminus of LEDGF/p75 and the DDE domain of pogZ, revealing an overlap in the binding of pogZ and HIV-1 integrase. Competition experiments showed that integrase is efficient in displacing pogZ from LEDGF/p75. Moreover, pogZ does not seem to play a role as a restriction factor of HIV. The finding that LEDGF/p75 is capable of interacting with a DDE domain protein that is not a lentiviral integrase points to a profound role of LEDGF/p75 in DDE domain protein function.

Molecular characterization and expression profile of a novel porcine gene differentially expressed in the muscle and backfat tissues from Chinese Meishan and Russian Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus dorsi muscle and backfat tissues from Chinese Meishan and Russian Large White pigs. One novel gene that was differentially expressed was identified through semi-quantitative RT-PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The cDNA sequence of this gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 402 amino acids that contains the putative conserved transposase DDE domain and further Blast analysis revealed that this protein has 100% homology with the Tn10 transposase from Oryza sativa, Serratia marcescens, and Salmonella, and therefore, this gene can be defined as the swine Tn10 transposase gene. This novel porcine gene was finally assigned to Gene ID: 100049649. The RT-PCR analysis of the tissue expression profile was carried out using the tissue cDNAs of one Meishan pig as the templates, and the result indicated that this novel swine gene is moderately expressed in fat, and weakly expressed in small intestine, liver, kidney, and spleen but almost not expressed in heart, ovary, muscle, and lung. Our experiment established the primary foundation for further research into the biological significance of swine Tn10 transposase gene.

Master: a novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.)

Members of a novel Master family of class II transposons were identified in the carrot genome. Two elements, 2.5 kb long DcMaster1 and 4.4 kb long DcMaster-a, are characterized by 22 bp imperfect terminal inverted repeats and by 3 bp target site duplications. GenBank search revealed that related elements are also present in Medicago truncatula, including a 5.1 kb element MtMaster-a. Both DcMaster-a and MtMaster-a contain open reading frames encoding for putative transposases with the complete DDE domain typical for plant class II transposable elements belonging to PIF/Harbinger superfamily, where the Master elements form a distinct group. Less than 10 copies of the DcMaster element containing the DDE domain are present in genomes of carrot and other Apiaceae, but more copies with internal deletions or insertions may occur. DcMaster elements were associated with putative coding regions in 8 of 14 identified insertion sites. PCR amplification of carrot genomic DNA using a primer complementary to TIRs of DcMaster gave products < 400 bp in size. We speculate that these may all represent a MITE-like family of transposable elements that we named Krak, present in the carrot genome in at least 3,600 copies.

Stable Gene Transfer and Expression in Human Primary T-Cells by the Sleeping Beauty Transposon System.

The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p<0.0001). In addition, a transposase mutant construct was incapable of mediating stable gene expression in human PBLs (n=6, p<0.0001), confirming that catalytic DDE domain is necessary for transposition in human primary T-cells. Immunophenotyping analysis in transposed T-cells showed that both CD4 and CD8 T-cells were transgene positive. SB-mediated high level of transgene expression in human T-cells was maintained in culture for at least 4 months without losing observable expression. Southern hybridization analysis showed a variety of transposon integrants among the 6 DsRed positive T-cell clones and no transposon sequences identifiable in the 2 DsRed negative clones. Sequencing of transposon:chromosome junctions in 5 out of 6 transposed T-cell clones confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably and functionally express a fusion protein consisting ofa surface receptor useful for positive T-cell selection anda “suicide” gene useful for elimination of transfected T-cells after chemotherapy.This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.

Characterisation of the luciferase gene and the 5' upstream region in the European glow-worm Lampyris noctiluca (Coleoptera: Lampyridae)

Beetle luciferase, a mono-oxygenase within the AMP-binding superfamily, is synthesized by bioluminescent beetles in concentrated levels within specialised cells clustering in the abdominal light organs. In vivo expression of luciferase has been rarely investigated and little is known about the role of enhancers and promoters in the expression of this gene. In order to investigate the gene structure and potential control of gene expression the luciferase gene along with 6 kb of upstream genomic sequence was char- acterised from the European glow-worm Lampyris noctiluca. Three TATA box motifs and a CAAT repeat were identified, two of these were found to be conserved in two other species of bioluminescent beetle. Although no enhancer regions were identified in the upstream sequence a region coding for a putative transposase DDE domain was identified 686 bp from the start codon of the lucife- rase gene. Although disrupted, the open reading frame also shared extensive identity to an mRNA transcript from the mosquito Anopheles gambiae. The remnants of an ancient transposase provide support for an ancestral luciferase transposition/insertion event that may have occurred within the genome of bioluminescent beetles.

Functional domains of the IS1 transposase: analysis in vivo and in vitro.

The IS1 bacterial insertion sequence family, considered to be restricted to Enterobacteria, has now been extended to other Eubacteria and to Archaebacteria, reviving interest in its study. To analyse the functional domains of the InsAB' transposase of IS1A, a representative of this family, we used an in vivo system which measures IS1-promoted rescue of a temperature-sensitive pSC101 plasmid by fusion with a pBR322::IS1 derivative. We also describe the partial purification of the IS1 transposase and the development of several in vitro assays for transposase activity. These included a DNA band shift assay, a transposase-mediated cleavage assay and an integration assay. Alignments of IS family members (http://www-is.biotoul.fr) not only confirmed the presence of an N-terminal helix-turn-helix and a C-terminal DDE motif in InsAB', but also revealed a putative N-terminal zinc finger. We have combined the in vitro and in vivo tests to carry out a functional analysis of InsAB' using a series of site-directed InsAB' mutants based on these alignments. The results demonstrate that appropriate mutations in the zinc finger and helix-turn-helix motifs result in loss of binding activity to the ends of IS1 whereas mutations in the DDE domain are affected in subsequent transposition steps but not in end binding.

Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum.

The transposable element impala is a member of the widespread superfamily of Tc1-mariner transposons, identified in the genome of the plant pathogenic fungus Fusarium oxysporum. This element is present in a low copy number and is actively transposed in the F. oxysporum strain F24 that is pathogenic for melons. The structure of the impala family was investigated by cloning and sequencing all the genomic copies. The analysis revealed that this family is composed of full-length and truncated copies. Four copies contained a long open reading frame that could potentially encode a transposase of 340 amino acids. The presence of conserved functional domains (a nuclear localisation signal, a catalytic DDE domain and a DNA-binding domain) suggests that these four copies may be autonomous elements. Sequence comparisons and phylogenetic analysis of the impala copies defined three subfamilies, which differ by a high level of nucleotide polymorphism (around 20%). The coexistence of these divergent subfamilies in the same genome may indicate that the impala family is of ancient origin and/or that it arose by successive horizontal transmission events.

The same two monomers within a MuA tetramer provide the DDE domains for the strand cleavage and strand transfer steps of transposition.

The chemistry of Mu transposition is executed within a tetrameric form of the Mu transposase (MuA protein). A triad of DDE (Asp, Asp35Glu motif) residues in the central domain of MuA (DDE domain) is essential for both the strand cleavage and strand transfer steps of transposition. Previous studies had suggested that complete Mu transposition requires all four subunits in the MuA tetramer to carry an active DDE domain. Using a mixture of MuA proteins with either wild-type or altered att-DNA binding specificities, we have now designed specific arrangements of MuA subunits carrying the DDE domain. From analysis of the abilities of oriented tetramers to carry out DNA cleavage and strand transfer from supercoiled DNA, a new picture of the disposition of DNA and protein partners during transposition has emerged. For DNA cleavage, two subunits of MuA located at attL1 and attR1 (sites that undergo cleavage) provide DDE residues in trans. The same two subunits contribute DDE residues for strand transfer, also in trans. Thus, only two active DDE+ monomers within the tetramer carry out complete Mu transposition. We also show that when the attR1-R2 arrangement used on supercoiled substrates is tested for cleavage on linear substrates, alternative chemically competent DNA-protein associations are produced, wherein the functional DDE subunits are positioned at R2 rather than at R1.