Human L1 retrotransposition: insights and peculiarities learned from a cultured cell retrotransposition assay

Long Interspersed Nuclear Elements (L1s or LINEs) are the most abundant retrotransposons in the human genome, and they comprise approximately 17% of DNA. L1 retrotransposition can be mutagenic, and deleterious insertions both in the germ-line and in somatic cells have resulted in disease. Recently, an assay was developed to monitor L1 retrotransposition in cultured human cells. This assay, for the first time, now allows for a systematic study of L1 retrotransposition at the molecular level. Here, I will review progress made in L1 biology during the past three years. In general, I will limit the discussion to studies conducted on human L1s. However, interesting parallels to rodent L1s and other non-LTR retrotransposons also will be discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

A versatile plasmid architecture for mammalian synthetic biology (VAMSyB)

Current molecular cloning strategies generally lack inter-compatibility, are not strictly modular, or are not applicable to engineer multi-gene expression vectors for transient and stable integration. A standardized molecular cloning platform would advance research, for example, by promoting exchange of vectors between groups. Here, we present a versatile plasmid architecture for mammalian synthetic biology, which we designate VAMSyB, consisting of a three-tier vector family. Tier-1 is designed for easy engineering of fusion constructs, as well as easy swapping of genes and modules to tune the functionality of the vector. Tier-2 is designed for transient multi-gene expression, and is constructed by directly transferring the engineered expression cassettes from tier-1 vectors. Tier-3 enables stable integration into a mammalian host cell through viral transduction, transposons, or homology-directed recombination via CRISPR. This VAMSyB architecture is expected to have broad applicability in the field of mammalian synthetic biology. The VAMSyB collection of plasmids will be available through Addgene.     

RNase L restricts the mobility of engineered retrotransposons in cultured human cells

Retrotransposons are mobile genetic elements, and their mobility can lead to genomic instability. Retrotransposon insertions are associated with a diverse range of sporadic diseases, including cancer. Thus, it is not a surprise that multiple host defense mechanisms suppress retrotransposition. The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a mechanism for restricting viral infections during the interferon antiviral response. Here, we investigated a potential role for the OAS-RNase L system in the restriction of retrotransposons. Expression of wild type (WT) and a constitutively active form of RNase L (NΔ385), but not a catalytically inactive RNase L mutant (R667A), impaired the mobility of engineered human LINE-1 (L1) and mouse intracisternal A-type particle retrotransposons in cultured human cells. Furthermore, WT RNase L, but not an inactive RNase L mutant (R667A), reduced L1 RNA levels and subsequent expression of the L1-encoded proteins (ORF1p and ORF2p). Consistently, confocal immunofluorescent microscopy demonstrated that WT RNase L, but not RNase L R667A, prevented formation of L1 cytoplasmic foci. Finally, siRNA-mediated depletion of endogenous RNase L in a human ovarian cancer cell line (Hey1b) increased the levels of L1 retrotransposition by ∼2-fold. Together, these data suggest that RNase L might function as a suppressor of structurally distinct retrotransposons.     

Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult-to-transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit-like self-inactivating lentiviral expression vector family which is compatible to state-of-the-art packaging and pseudotyping technologies and contains, besides essential cis-acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate-early promoter (P_hCMV) or the human elongation factor 1α (P_hEF1α), (iii) P_hCMV– or P_PGK– (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein-based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size-optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5′U3, R-U5-ψ+-SD, cPPT-RRE-SA and 3′LTR_ΔU3 modules or placed within the 5′U3 (–78 bp) and 3′LTR_ΔU3 (8666 bp). pLentiModule enables straightforward cassette-type module swapping between lentiviral expression vector family members and facilitates the design of Tat-independent (replacement of 5′LTR by heterologous promoter elements), regulated and self-excisable proviruses (insertion of responsive operators or LoxP in the 3′LTR_ΔU3 element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi-purpose construction kit-like vector series described here is compatible with itself as well as many other (non-viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human diseases.     

Development of New Modular Genetic Tools for Engineering the Halophilic Archaeon Halobacterium salinarum

Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i) the E. coli’s specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii) the resistance marker and (iii) the replication origin, which are specific to H. salinarum and (iv) the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.     

Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.     

Interspersed repeats are found predominantly in the "old" alpha satellite families

The biased distribution of dispersed repeat insertions in various types of primate specific α satellites (AS) is being discussed in the literature in relation to the modes of AS evolution and their possible roles in maintenance and disruption of functional centromeres. However, such a bias has not been properly documented on a genome-wide scale so far. In this work, using a representative sample of about 100 insertions we show that the “old” AS contains at least 10 times more dispersed repeats than the “new” one. In the new arrays insertions accumulate mostly in poorly homogenized areas, presumably in the edges, and in the old AS, throughout the whole array length. Dating of L1 insertions in the old AS revealed that their massive accumulation started at or after the time when the new AS emerged and expanded in the genome and the centromere function had shifted to the new AS arrays.     

Rapid gene cloning using terminator primers and modular vectors

We seek to create useful biological diversity by exploiting the modular nature of genetic information. In this report we describe experiments that focus on the modular nature of plasmid cloning vectors. Bacterial plasmids are modular entities composed of origins of replication, selectable markers and other components. We describe a new ligation-independent cloning method that allows for rapid and seamless assembly of vectors from component modules. We further demonstrate that gene cloning can be accomplished simultaneously with assembly of a modular vector. This approach provides considerable flexibility as it allows for ‘menu driven’ cloning of genes into custom assembled modular vectors.     

Selection against LINE-1 retrotransposons results principally from their ability to mediate ectopic recombination

LINE-1 (L1) retrotransposons constitute the most successful family of autonomous retroelements in mammals and they represent at least 17% of the size of the human genome. L1 insertions have occasionally been recruited to perform a beneficial function but the vast majority of L1 inserts are either neutral or deleterious. The basis for the deleterious effect of L1 remains a matter of debate and three possible mechanisms have been suggested: the direct effect of L1 inserts on gene activity, genetic rearrangements caused by L1-mediated ectopic recombination, or the retrotransposition process per se. We performed a genome-wide analysis of the distribution of L1 retrotransposons relative to the local recombination rate and the age and length of the elements. The proportion of L1 elements that are longer than 1.2 Kb is higher in low-recombining regions of the genome than in regions with a high recombination rate, but the genomic distributions of full-length elements (i.e. elements capable of retrotransposition) and long truncated elements were indistinguishable. We also found that the intensity of selection against long elements is proportional to the replicative success of L1 families. This suggests that the deleterious effect of L1 elements results principally from their ability to mediate ectopic recombination.     

裸燕麦Ty1-copia类反转录转座子反转录酶序列的分离、鉴定与分析

本研究根据Ty1-copia类反转录转座子反转录酶的保守区设计简并引物,通过PCR扩增,从裸燕麦(Avena nuda L.)品种‘品燕1号’基因组中分离获得23条Ty1-copia类反转录转座子序列,并对序列特征、系统发育关系及其转录活性进行分析。结果显示,23条Ty1-copia类反转录转座子存在较高的异质性,序列间的一致性为45%～98%,存在插入、移码和终止密码突变,但频率不高;系统发育分析结果表明,燕麦Ty1-copia类反转录转座子在进化过程中主要为垂直传递。本研究通过检索燕麦基因表达数据库,发现了5个有转录活性的Ty1-copia类反转录转座子。     

The non-autonomous retrotransposon SVA is trans-mobilized by the human LINE-1 protein machinery

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.     

Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition

LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic ‘parasites’.     

A modified pET vector that augments heterologous protein expression

Summary We describe a modification which can be carried out on the pET vector series that destroys the rop function in these vectors. The modified vectors have the following advantages: i) increased plasmid copy number, ii) augmented heterologous protein expression of the target gene, iii) elimination of plasmid incompatibility, and iv) they are smaller in size, consequently, they have higher transformation efficiency and can accommodate larger DNA inserts. Additionally, several restriction sites become unique allowing easier restriction mapping of inserts when required.     

Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus‐like particle

Virus‐like particles (VLPs) and capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of capsomeres, and VLP assembly. In this study, three module designs of a rotavirus hydrophobic peptide (RV10) were synthesized using synthetic biology. Among the three synthetic modules, modularization of the murine polyomavirus VP1 with a single copy of RV10 flanked by long linkers and charged residues resulted in the expression of stable modular capsomeres. Further employing the approach of module titration of RV10 modules on each capsomere via Escherichia coli co‐expression of unmodified VP1 and modular VP1‐RV10 successfully translated purified modular capomeres into modular VLPs when assembled in vitro. Our results demonstrate that tailoring the physicochemical properties of modules to enhance modular capsomeres stability is achievable through synthetic biology designs. Combined with module titration strategy to avoid steric hindrance to intercapsomere interactions, this allows bioprocessing of bacterially produced in vitro assembled modular VLPs.     

Mutagenesis in rodents using the L1 retrotransposon

LINE1 (L1) retrotransposons are genetic elements that are present in all mammalian genomes. L1s are active in both humans and mice, and are capable of copying themselves and inserting the copy into a new genomic location. These de novo insertions occasionally result in disease. Endogenous L1 retrotransposons can be modified to increase their activity and mutagenic power in a variety of ways. Here we outline the advantages of using modified L1 retrotransposons for performing random mutagenesis in rodents and discuss several potential applications.     

Modularity of serpins. A bifunctional chimera possessing alpha1-proteinase inhibitor and thyroxine-binding globulin properties.

An exciting application of protein engineering is the creation of proteins with novel functions by the retrofitting of native proteins. Such attempts might be facilitated by the idea of a mosaic architecture of proteins out of structural units. Even though numerous theoretical concepts deal with the delineation of structural "modules," their potential in the design of proteins has not yet been sufficiently exploited. To address this question we used a gain of function approach by designing modular chimeric molecules out of two structurally homologous but functionally diverse members of the superfamily of serine-proteinase inhibitors, alpha1-proteinase inhibitor and thyroxine-binding globulin. Substitution of two of four alpha1-proteinase inhibitor modules (Lys222 to Leu288 and Pro362 to Lys394, respectively), identified by alpha-backbone distance analysis, with their thyroxine-binding globulin homologues resulted in a bifunctional chimera with inhibition of human leukocyte elastase and high affinity thyroxine binding. To our knowledge, this is the first report on a bifunctional chimera engineered from modules of homologous globular proteins. Our results demonstrate how a modular concept can facilitate the design of new functional proteins by swapping structural units chosen from members of a protein superfamily.     

Mos1-mediated insertional mutagenesis in Caenorhabditis elegans

We describe a protocol for mutating genes in the nematode Caenorhabditis elegans using the Mos1 transposon of Drosophila mauritiana. Mutated genes containing a Mos1 insertion are molecularly tagged by this heterologous transposable element. Mos1 insertions can therefore be identified in as little as 3 weeks using only basic molecular biology techniques. Mutagenic efficiency of Mos1 is tenfold lower than classical chemical mutagens. However, the ease and speed with which mutagenic insertions can be mapped compares favorably with the vast amount of work involved in classical genetic mapping. Therefore, Mos1 could be the tool of choice when screening procedures are efficient. In addition, Mos1 mutagenesis can greatly simplify the mapping of mutations that exhibit low penetrance, subtle or synthetic phenotypes. The recent development of targeted engineering of C. elegans loci carrying Mos1 insertions further increases the attractiveness of Mos1-mediated mutagenesis.