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Abstract

Expression vectors (EVs) are artificial nucleic acid molecules with a modular structure that allows for the transcription of DNA sequences of interest in either cellular or cell-free environments. These vectors have emerged as cross-disciplinary tools with multiple applications in an expanding Life Sciences market. The cis-regulatory sequences (CRSs) that control the transcription in EVs are typically sourced from either viruses or from characterized genes. However, the recent advancement in transposable elements (TEs) technology provides attractive alternatives that may enable a significant improvement in the design of EVs. Commonly known as "jumping genes," due to their ability to move between genetic loci, TEs are constitutive components of both eukaryotic and prokaryotic genomes. TEs harbor native CRSs that allow the regulated transcription of transposition-related genes. However, some TE-related CRSs display striking characteristics, which provides the opportunity to reconsider TEs as lead actors in the design of EVs. In this article, we provide a synopsis of the transcriptional control elements commonly found in EVs together with an extensive discussion of their advantages and limitations. We also highlight the latest findings that may allow for the implementation of TE-derived sequences in the EVs feasible, possibly improving existing vectors. By introducing this new concept of TEs as a source of regulatory sequences, we aim to stimulate a profitable discussion of the potential advantages and benefits of developing a new generation of EVs based on the use of TE-derived control sequences.

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... Initially regarded as "junk" and "useless", TEs turned out to be considered as evolutionarily flagships after reconsidering the role they have had and still have in shaping genomes and their functioning. Moreover, the characterization of many transposition systems has led to the development of efficient DNA integration tools [1] as well as powerful genome engineering systems [2,3], and to the implementation of TE control regions into efficient expression systems [4]. ...
... Most known TEs have limited transposition performances (i.e., low transposition rate, narrow host specificity) or they have low flexibility (i.e., they are too large and complex or display low cargo capability) to allow the development of efficient genome integration systems. Nevertheless, many TEs are studied for their role in shaping genome structure [12] and gene expression [13] or to develop new and alternative technologies [3,4,14,15]. ...
... It also suggests a common origin of this promoter type, and a possible common survival strategy for the elements belonging to the Tc1/mariner superfamily [105]. The discovery of the blurry promoters in the Bari family has also opened the possibility to implement them, together with many other TE-derived transcriptional control sequences, into expression modules in order to expand the repertoire of the existing expression vectors with a considerable improvement of their performances [4]. The % activity compared to strong species-specific constitutive promoters (SV40, copia, URA3, and CAT for human, fly, yeast, and bacteria cells, respectively), arbitrarily assumed as 100%, is reported on Y axis of the chart. ...
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Transposable elements (TEs) have been historically depicted as detrimental genetic entities that selfishly aim at perpetuating themselves, invading genomes, and destroying genes. Scientists often co-opt “special” TEs to develop new and powerful genetic tools, that will hopefully aid in changing the future of the human being. However, many TEs are gentle, rarely unleash themselves to harm the genome, and bashfully contribute to generating diversity and novelty in the genomes they have colonized, yet they offer the opportunity to develop new molecular tools. In this review we summarize 30 years of research focused on the Bari transposons. Bari is a “normal” transposon family that has colonized the genomes of several Drosophila species and introduced genomic novelties in the melanogaster species. We discuss how these results have contributed to advance the field of TE research and what future studies can still add to the current knowledge.
... As a consequence of the biological significance of TEs, TEs have recently been used as an integration tool in fundamental research [21] and in gene therapy [22]. TEs, or parts thereof, can also be implemented into common molecular biology tools, such as expression vectors [23]. In addition, TEs have been suggested as new markers (together with mitochondrial polymorphisms and Y-chromosome polymorphisms) to describe the evolutionary history of a species, or even of single individuals [24,25]. ...
... Both class I and class II TEs have autonomous (containing open reading frames, ORFs) and non-autonomous (absence of encoding potential while lacking transposition ability) TEs [12,23,60,61]. Class II autonomous TEs can encode transposase and helicase enzymes for cut-and-paste mechanisms [62]. ...
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Plant development processes are regulated by epigenetic alterations that shape nuclear structure, gene expression, and phenotypic plasticity; these alterations can provide the plant with protection from environmental stresses. During plant growth and development, these processes play a significant role in regulating gene expression to remodel chromatin structure. These epigenetic alterations are mainly regulated by transposable elements (TEs) whose abundance in plant genomes results in their interaction with genomes. Thus, TEs are the main source of epigenetic changes and form a substantial part of the plant genome. Furthermore, TEs can be activated under stress conditions, and activated elements cause mutagenic effects and substantial genetic variability. This introduces novel gene functions and structural variation in the insertion sites and primarily contributes to epigenetic modifications. Altogether, these modifications indirectly or directly provide the ability to withstand environmental stresses. In recent years, many studies have shown that TE methylation plays a major role in the evolution of the plant genome through epigenetic process that regulate gene imprinting, thereby upholding genome stability. The induced genetic rearrangements and insertions of mobile genetic elements in regions of active euchromatin contribute to genome alteration, leading to genomic stress. These TE-mediated epigenetic modifications lead to phenotypic diversity, genetic variation, and environmental stress tolerance. Thus, TE methylation is essential for plant evolution and stress adaptation, and TEs hold a relevant military position in the plant genome. High-throughput techniques have greatly advanced the understanding of TE-mediated gene expression and its associations with genome methylation and suggest that controlled mobilization of TEs could be used for crop breeding. However, development application in this area has been limited, and an integrated view of TE function and subsequent processes is lacking. In this review, we explore the enormous diversity and likely functions of the TE repertoire in adaptive evolution and discuss some recent examples of how TEs impact gene expression in plant development and stress adaptation.
... The cut-and-paste transposition reaction requires the recognition of two TIRs by the transposase, to cleave and release the DNA transposon from the original site, followed by the integration of the excised transposon into a new location, executed by the same transposase (6). Because of their inherent capacity for insertion into DNA, cut-andpaste transposons can be developed into powerful tools for genome manipulations, such as gene discovery applications in functional genomics and gene delivery systems for transgenesis and gene therapy (7)(8)(9). Cut-and-paste transposons, in particular, have demonstrated great potential as delivery vectors in germline transgenesis and insertional mutagenesis (7,8), with ZB displaying an efficient enhancer trapping in zebrafish and mice (10). Furthermore, the application of cut-and-paste transposons in gene therapy has been extensively reported and shows great potential in cancer immune therapy (11)(12)(13). ...
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The discovery of new, active DNA transposons can expand the range of genetic tools and provide more options for genomic manipulation. In this study, a bioinformatics analysis suggested that Passer (PS) transposons, which are members of the pogo superfamily, show signs of recent and current activity in animals and may be active in some species. Cell-based transposition assays revealed that the native PS transposases from Gasterosteus aculeatus and Danio rerio displayed very high activity in human cells relative to the Sleeping Beauty transposon. A typical overproduction inhibition phenomenon was observed for PS, and transposition capacity was decreased by ∼12% with each kilobase increase in the insertion size. Furthermore, PS exhibited a pronounced integration preference for genes and their transcriptional regulatory regions. We further show that two domesticated human proteins derived from PS transposases have lost their transposition activity. Overall, PS may represent an alternative with a potentially efficient genetic manipulation tool for transgenesis and mutagenesis applications.
... Transposable elements are abundant in all forms of life, but the active transposable elements are a hotspot for genomic research, as they can jump and actively multiply their numbers [44]. The transcriptomic analysis of the S. chinensis genome indicates that many TEs of different lineages are transcriptionally active. ...
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menu Journals IJMS Volume 23 Issue 24 10.3390/ijms232415967 settingsOrder Article Reprints Open AccessArticle Mobilome of the Rhus Gall Aphid Schlechtendalia chinensis Provides Insight into TE Insertion-Related Inactivation of Functional Genes by Aftab Ahmad andZhumei Ren * School of Life Science, Shanxi University, Taiyuan 030006, China * Author to whom correspondence should be addressed. Int. J. Mol. Sci. 2022, 23(24), 15967; https://doi.org/10.3390/ijms232415967 Received: 29 October 2022 / Revised: 7 December 2022 / Accepted: 12 December 2022 / Published: 15 December 2022 (This article belongs to the Section Molecular Genetics and Genomics) Download Browse Figures Review Reports Versions Notes Abstract Transposable elements (TEs) comprise a considerable proportion of insect genomic DNA; how they contribute to genome structure and organization is still poorly understood. Here, we present an analysis of the TE repertoire in the chromosome-level genome assembly of Rhus gall aphid Schlechtendalia chinensis. The TE fractions are composed of at least 32 different superfamilies and many TEs from different families were transcriptionally active in the S. chinensis genome. Furthermore, different types of transposase-derived proteins were also found in the S. chinensis genome. We also provide insight into the TEs related insertional inactivation, and exogenization of TEs in functional genes. We considered that the presence of TE fragments in the introns of functional genes could impact the activity of functional genes, and a large number of TE fragments in introns could lead to the indirect inactivation of functional genes. The present study will be beneficial in understanding the role and impact of TEs in genomic evolution of their hosts.
... Cyclin-dependent kinase 4/6 inhibitors (CKD4/6i) selectively target proliferating cancer cells and can be an excellent therapeutic strategy to enhance or boost the immune signaling in cancer cells without hampering normal cells. Another future application of TEs is in the recombinant DNA technology (rDT), as it is estimated that the global rDT market will reach 850 billion USD by 2025, of which TE-based expression vectors will be of a substantial proportion [209]. Besides these, application of TEs in basic as well as applied biological sciences is in offing (Figure 4), transforming "foes into friends". ...
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Most living organisms have in their genome a sizable proportion of DNA sequences capable of mobilization; these sequences are commonly referred to as transposons, transposable elements (TEs), or jumping genes. Although long thought to have no biological significance, advances in DNA sequencing and analytical technologies have enabled precise characterization of TEs and confirmed their ubiquitous presence across all forms of life. These findings have ignited intense debates over their biological significance. The available evidence now supports the notion that TEs exert major influence over many biological aspects of organismal life. Transposable elements contribute significantly to the evolution of the genome by giving rise to genetic variations in both active and passive modes. Due to their intrinsic nature of mobility within the genome, TEs primarily cause gene disruption and large-scale genomic alterations including inversions, deletions, and duplications. Besides genomic instability, growing evidence also points to many physiologically important functions of TEs, such as gene regulation through cis-acting control elements and modulation of the transcriptome through epigenetic control. In this review, we discuss the latest evidence demonstrating the impact of TEs on genome stability and the underling mechanisms, including those developed to mitigate the deleterious impact of TEs on genomic stability and human health. We have also highlighted the potential therapeutic application of TEs.
... Many cis-regulatory elements in TEs evolved to have similar activity in multiple diverse species, making TEs a promising source for the development of new expression vectors in biotechnology [223]. ...
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Transposable elements (TEs) have been extensively studied for decades. In recent years, the introduction of whole-genome and whole-transcriptome approaches, as well as single-cell resolution techniques, provided a breakthrough that uncovered TE involvement in host gene expression regulation underlying multiple normal and pathological processes. Of particular interest is increased TE activity in neuronal tissue, and specifically in the hippocampus, that was repeatedly demonstrated in multiple experiments. On the other hand, numerous neuropathologies are associated with TE dysregulation. Here, we provide a comprehensive review of literature about the role of TEs in neurons published over the last three decades. The first chapter of the present review describes known mechanisms of TE interaction with host genomes in general, with the focus on mammalian and human TEs; the second chapter provides examples of TE exaptation in normal neuronal tissue, including TE involvement in neuronal differentiation and plasticity; and the last chapter lists TE-related neuropathologies. We sought to provide specific molecular mechanisms of TE involvement in neuron-specific processes whenever possible; however, in many cases, only phenomenological reports were available. This underscores the importance of further studies in this area.
... mobile self-regulatory activity, TEs have many biotechnological implications. They can be used as an expression vector and a vector in gene therapy, e.g., sleeping beauty transposon system [7,8]. ...
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Horizontal transfer of transposons (HTT) is an essential source of genomic evolution in eukaryotes. The HTT dynamics are well characterized in eukaryotes, including insects; however, there is a considerable gap in knowledge about HTT regarding many eukaryotes’ species. In this study, we analyzed the events of the HTT between Rhus gall aphids (Hemiptera) and other insects. We analyzed the Mariner-like transposable elements (MLEs) belonging to Rhus gall aphids for the possible HT events. The MLEs have a patchy distribution and high similarity over the entire element length with insect MLEs from different orders. We selected representative sequences from the Rhus gall MLEs and identified five events of HT between MLEs of Rhus gall aphids and other insects from five different orders. We also found multiple HTT events among the MLEs of insects from the five orders, demonstrating that these Mariner elements have been involved in recurrent HT between Rhus gall aphids and other insects. Our current study closed the knowledge gap surrounding HTT and reported the events between Rhus gall aphids and other insects for the first time. We believe that this study about HTT events will help us understand the evolution and spread of transposable elements in the genomes of Rhus gall aphids.
... TEs are known as "jumping" DNA sequences allowing several insertions into host genomes and generating multiple sites for chromosomal rearrangements, which may have either deleterious or beneficial consequences [8][9][10]. Furthermore, TEs can confer selective advantages through their insertion sites by either enhancing or suppressing gene expression or being domesticated as a new host gene [11][12][13][14]. Several studies showed that TEs insertions into specific genes were linked to insecticide resistance. ...
Article
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Transposable elements (TEs) are genetically mobile units that move from one site to another within a genome. These units can mediate regulatory changes that can result in massive changes in genes expression. In fact, a precise identification of TEs can allow the detection of the mechanisms involving these elements in gene regulation and genome evolution. In the present study, a genome-wide analysis of the Hemipteran pest Bemisia tabaci was conducted using bioinformatics tools to identify, annotate and estimate the age of TEs, in addition to their insertion sites, within or near of the defensome genes involved in insecticide resistance. Overall, 1,292,393 TE copies were identified in the B. tabaci genome grouped into 4872 lineages. A total of 699 lineages were found to belong to Class I of TEs, 1348 belong to Class II, and 2825 were uncategorized and form the largest part of TEs (28.81%). The TE age estimation revealed that the oldest TEs invasion happened 14 million years ago (MYA) and the most recent occurred 0.2 MYA with the insertion of Class II TE elements. The analysis of TE insertion sites in defensome genes revealed 94 insertions. Six of these TE insertions were found within or near previously identified differentially expressed insecticide resistance genes. These insertions may have a potential role in the observed insecticide resistance in these pests.
... Indeed, depending on the target and mode of their transposition and recombination, mobile elements can be exapted to new cis-regulatory elements (Britten, 1996;Marino-Ramirez et al., 2005), disrupt or rewire regulatory networks (Feschotte, 2008;Moschetti et al., 2020;Sundaram and Wysocka, 2020), and cause chromosomal-level rearrangements (Gray, 2000). Besides, TEs are important tools for the development of new genomic integration (Sandoval-Villegas et al., 2021) and expression vector technologies (Palazzo and Marsano, 2021). TEs are present in every eukaryotic genome in very different proportions and classes (Wells and Feschotte, 2020), with both random drift and natural selection contributing to their differential amplification in divergent lineages (Lynch and Conery, 2003;Kent et al., 2017). ...
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In comparison with other molluscs and bilaterians, the genomes of coleoid cephalopods (squid, cuttlefish, and octopus) sequenced so far show remarkably different genomic organization that presumably marked the early evolution of this taxon. The main driver behind this genomic rearrangement remains unclear. About half of the genome content in coleoids is known to consist of repeat elements; since selfish DNA is one of the powerful drivers of genome evolution, its pervasiveness could be intertwined with the emergence of cephalopod-specific genomic signatures and could have played an important role in the reorganization of the cephalopod genome architecture. However, due to abundant species-specific repeat expansions, it has not been possible so far to identify the ancient shared set of repeats associated with coleoid divergence. By means of an extensive repeat element re-evaluation and annotation combined with network sequence divergence approaches, we are able to identify and characterize the ancient repeat complement shared by at least four coleoid cephalopod species. Surprisingly, instead of the most abundant elements present in extant genomes, lower-copy-number DNA and retroelements were most associated with ancient coleoid radiation. Furthermore, evolutionary analysis of some of the most abundant families shared in Octopus bimaculoides and Euprymna scolopes disclosed within-family patterns of large species-specific expansions while also identifying a smaller shared expansion in the coleoid ancestor. Our study thus reveals the apomorphic nature of retroelement expansion in octopus and a conserved complement composed of several DNA element types and fewer LINE families.
... Furthermore, TEs have become powerful tools for transgene integration and mutagenesis [8,9]. They are also progressively acquiring credit as gene therapy vectors [10,11] and as sources of ectopic gene expression tools [12]. ...
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Transposable elements (TEs) are abundant components of constitutive heterochromatin of the most diverse evolutionarily distant organisms. TEs enrichment in constitutive heterochromatin was originally described in the model organism Drosophila melanogaster, but it is now considered as a general feature of this peculiar portion of the genomes. The phenomenon of TE enrichment in constitutive heterochromatin has been proposed to be the consequence of a progressive accumulation of transposable elements caused by both reduced recombination and lack of functional genes in constitutive heterochromatin. However, this view does not take into account classical genetics studies and most recent evidence derived by genomic analyses of heterochromatin in Drosophila and other species. In particular, the lack of functional genes does not seem to be any more a general feature of heterochromatin. Sequencing and annotation of Drosophila melanogaster constitutive heterochromatin have shown that this peculiar genomic compartment contains hundreds of transcriptionally active genes, generally larger in size than that of euchromatic ones. Together, these genes occupy a significant fraction of the genomic territory of heterochromatin. Moreover, transposable elements have been suggested to drive the formation of heterochromatin by recruiting HP1 and repressive chromatin marks. In addition, there are several pieces of evidence that transposable elements accumulation in the heterochromatin might be important for centromere and telomere structure. Thus, there may be more complexity to the relationship between transposable elements and constitutive heterochromatin, in that different forces could drive the dynamic of this phenomenon. Among those forces, preferential transposition may be an important factor. In this article, we present an overview of experimental findings showing cases of transposon enrichment into the heterochromatin and their positive evolutionary interactions with an impact to host genomes.
... Our knowledge on the mechanisms of how gene expressions are altered in mothers is limited. An intriguing possibility is regulation by transposable elements, which can carry potent transcriptional cis-regulators that could recruit specific transcription factors, even in a tissue-specific manner [40,41]. There is available evidence of the involvement of transposable elements insertions in gene expression in the brain [42][43][44] and their role in the zebra finch brain is also plausible. ...
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(1) Background: The objective of this study was to uncover genomic causes of parental care. Since birds do not lactate and, therefore, do not show the gene expressional changes required for lactation, we investigate gene expression associated with parenting in caring and non-caring females in an avian species, the small passerine bird zebra finch (Taeniopygia guttata). Here, we compare expression patterns in the hypothalamic–septal region since, previously, we showed that this area is activated in parenting females. (2) Methods: Transcriptome sequencing was first applied in a dissected part of the zebra finch brain related to taking care of the nestlings as compared to a control group of social pairs without nestlings. (3) Results: We found genes differentially expressed between caring and non-caring females. When introducing a log2fold change threshold of 1.5, 13 annotated genes were significantly upregulated in breeding pairs, while 39 annotated genes were downregulated. Significant enrichments of dopamine and acetylcholine biosynthetic processes were identified among upregulated pathways, while pro-opiomelanocortin and thyroid hormone pathways were downregulated, suggesting the importance of these systems in parental care. Network analysis further suggested neuro-immunological changes in mothers. (4) Conclusions: The results confirm the roles of several hypothesized major pathways in parental care, whereas novel pathways are also proposed.
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In recent decades, experimental data has accumulated indicating that short interspersed nuclear elements (SINEs) can play a significant functional role in the regulation of gene expression in the host genome. In addition, molecular markers based on SINE insertion polymorphisms have been developed and are widely used for genetic differentiation of populations of eukaryotic organisms. Using routine bioinformatics analysis and publicly available genomic DNA and small RNA-seq data, we first described nine SINEs in the genome of the German cockroach, Blattella germanica . All described SINEs have tRNA promoters, and the start of their transcription begins 11 bp upstream of an “A” box of these promoters. The number of copies of the described SINEs in the B . germanica genome ranges from several copies to more than a thousand copies in a SINE-specific manner. Some of the described SINEs and their degenerate copies can be localized both in the introns of genes and loci known as piRNA clusters. piRNAs originating from piRNA clusters are shown to be mapped to seven of the nine types of SINEs described, including copies of SINEs localized in gene introns. We speculate that SINEs, localized in the introns of certain genes, may regulate the level of expression of these genes by a PIWI-related molecular mechanism.
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Mobility of eukaryotic transposable elements (TEs) are finely regulated to avoid an excessive mutational load caused by their movement. The transposition of retrotransposons is usually regulated through the interaction of host-and TE-encoded proteins, with non-coding regions (LTR and 5′-UTR) of the transposon. Examples of new potent cis-acting sequences, identified and characterized in the non-coding regions of retrotransposons, include the insulator of gypsy and Idefix, and the enhancer of ZAM of Drosophila melanogaster. Recently we have shown that in the 5′-UTR of the LTR-retrotransposon ZAM there is a sequence structured in tandem-repeat capable of operating as an insulator both in Drosophila (S2R +) and human cells (HEK293). Here, we test the hypothesis that tandem repeated 5′-UTR of a different LTR-retrotransposon could accommodate similar regulatory elements. The comparison of the 5′-UTR of some LTR-transposons allowed us to identify a shared motif of 13bp, called Transposable Element Redundant Motif (TERM). Surprisingly, we demonstrated, by Yeast One-Hybrid assay, that TERM interacts with the D. melanogaster ribosomal protein RpL22. The Drosophila RpL22 has additional Ala-, Lys-and Pro-rich sequences at the amino terminus, which resembles the carboxy-terminal portion of histone H1 and histone H5. For this reason, it has been hypothesized that RpL22 might have two functions, namely the role in organizing the ribosome, and a potential regulatory role involving DNA-binding similar to histone H1, which represses transcription in Drosophila. In this paper, we show, by two independent sets of experiments, that DmRpL22 is able to directly and specifically bind DNA of Drosophila melanogaster.
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Background Transposable elements (TEs) are a significant component of eukaryotic genomes and play essential roles in genome evolution. Mounting evidence indicates that TEs are highly transcribed in early embryo development and contribute to distinct biological functions and tissue morphology. Results We examine the epigenetic dynamics of mouse TEs during the development of five tissues: intestine, liver, lung, stomach, and kidney. We found that TEs are associated with over 20% of open chromatin regions during development. Close to half of these accessible TEs are only activated in a single tissue and a specific developmental stage. Most accessible TEs are rodent-specific. Across these five tissues, 453 accessible TEs are found to create the transcription start sites of downstream genes in mouse, including 117 protein-coding genes and 144 lincRNA genes, 93.7% of which are mouse-specific. Species-specific TE-derived transcription start sites are found to drive the expression of tissue-specific genes and change their tissue-specific expression patterns during evolution. Conclusion Our results suggest that TE insertions increase the regulatory potential of the genome, and some TEs have been domesticated to become a crucial component of gene and regulate tissue-specific expression during mouse tissue development.
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Transposable elements (TEs) are constitutive components of both eukaryotic and prokaryotic genomes. The role of TEs in the evolution of genes and genomes has been widely assessed over the past years in a variety of model and non-model organisms. Drosophila is undoubtedly among the most powerful model organisms used for the purpose of studying the role of transposons and their effects on the stability and evolution of genes and genomes. Besides their most intuitive role as insertional mutagens, TEs can modify the transcriptional pattern of host genes by juxtaposing new cis-regulatory sequences. A key element of TE biology is that they carry transcriptional control elements that fine-tune the transcription of their own genes, but that can also perturb the transcriptional activity of neighboring host genes. From this perspective, the transposition-mediated modulation of gene expression is an important issue for the short-term adaptation of physiological functions to the environmental changes, and for long-term evolutionary changes. Here, we review the current literature concerning the regulatory and structural elements operating in cis provided by TEs in Drosophila. Furthermore, we highlight that, besides their influence on both TEs and host genes expression, they can affect the chromatin structure and epigenetic status as well as both the chromosome’s structure and stability. It emerges that Drosophila is a good model organism to study the effect of TE-linked regulatory sequences, and it could help future studies on TE–host interactions in any complex eukaryotic genome.
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During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, -1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation.
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Inducible gene expression systems are favored over stable expression systems in a wide variety of basic and applied research areas, including functional genomics, gene therapy, tissue engineering, biopharmaceutical protein production and drug discovery. This is because they are mostly reversible and thus more flexible to use. Furthermore, compared to constitutive expression, they generally exhibit a higher efficiency and have fewer side effects, such as cell death and delayed growth or development. Empowered by decades of development of inducible gene expression systems, researchers can now efficiently activate or suppress any gene, temporarily and quantitively at will, depending on experimental requirements and designs. Here, we review a number of most commonly used mammalian inducible expression systems and provide basic standards and criteria for the selection of the most suitable one.
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Most genomes are populated by hundreds of thousands of sequences originated from mobile elements. On the one hand, these sequences present a real challenge in the process of genome analysis and annotation. On the other hand, they are very interesting biological subjects involved in many cellular processes. Here we present an overview of transposable elements biodiversity, and we discuss different approaches to transposable elements detection and analyses.
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In the past years, the demand for small batches of clinical grade plasmid DNA has been growing. For that purpose, we designed and qualified a scaled-down Good Manufacturing Practices (GMP) production method, able to produce small batches (1-4 mg) of plasmid. The developed method does not require any complex production equipment and utilizes only disposable production materials, which makes it easy to implement and simplifies line-clearance. We have successfully used this method to produce several small batches of two different plasmids. The produced plasmids, both formulated in an Electroporation Buffer, are mixed and filled into small, single-use, aliquots. Quality control confirmed the robustness of the developed method and a stability study showed that the final formulation is stable for at least two years. The final patient formulation will be subsequently used in a phase I/II clinical trial in which retina cells of patients with Age Related Macular Degeneration, are transfected. The presented production method can be generically used for other plasmid constructs and final formulation designs.
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Transposable elements (TEs) are thought to have helped establish gene regulatory networks. Both the embryonic and extraembryonic lineages of the early mouse embryo have seemingly co-opted TEs as enhancers, but there is little evidence that they play significant roles in gene regulation. Here we tested a set of long terminal repeat TE families for roles as enhancers in mouse embryonic and trophoblast stem cells. Epigenomic and transcriptomic data suggested that a large number of TEs helped to establish tissue-specific gene expression programmes. Genetic editing of individual TEs confirmed a subset of these regulatory relationships. However, a wider survey via CRISPR interference of RLTR13D6 elements in embryonic stem cells revealed that only a minority play significant roles in gene regulation. Our results suggest that a subset of TEs are important for gene regulation in early mouse development, and highlight the importance of functional experiments when evaluating gene regulatory roles of TEs.
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Background We have recently described a peculiar feature of the promoters in two Drosophila Tc1-like elements, Bari1 and Bari3. The AT-richness and the presence of weak core-promoter motifs make these promoters, that we have defined “blurry”, able to activate transcription of a reporter gene in cellular systems as diverse as fly, human, yeast and bacteria. In order to clarify whether the blurry promoter is a specific feature of the Bari transposon family, we have extended this study to promoters isolated from three additional DNA transposon and from two additional LTR retrotransposons. Results Here we show that the blurry promoter is also a feature of two vertebrate transposable elements, Sleeping Beauty and Hsmar1, belonging to the Tc1/mariner superfamily. In contrast, this feature is not shared by the promoter of the hobo transposon, which belongs to the hAT superfamily, nor by LTR retrotransposon-derived promoters, which, in general, do not activate transcription when introduced into non-related genomes. Conclusions Our results suggest that the blurry promoter could be a shared feature of the members of the Tc1/mariner superfamily with possible evolutionary and biotechnological implications. Electronic supplementary material The online version of this article (10.1186/s13100-019-0155-6) contains supplementary material, which is available to authorized users.
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Transposable elements (TEs) are major components of the human genome constituting at least half of it. More than half a century ago, Barbara McClintock and later Roy Britten and Eric Davidson have postulated that they might be major players in the host gene regulation. We have scanned a large amount of data produced by ENCODE project for active transcription binding sites (TFBSs) located in TE-originated parts of polymerase II promoters. In total, more than 35,000 promoters in six different tissues were analyzed and over 26,000 of them harbored TEs. Moreover, these TEs usually provide one or more of TFBSs in the host promoters, which resulted in more than 6% of active TFBSs in these regions located in the TE-originated sequences. Rewiring of transcription circuits played a major role in mammalian evolution and consequently increased their functional and morphological diversity. In this large-scale analysis, we have demonstrated that TEs contributed a large fraction of human TFBSs. Interestingly, these TFBSs usually act in a tissue-specific manner. Thus, our study clearly showed that TEs played a significant role in shaping expression pattern in mammals and humans in particular. Furthermore, since several TE families are still active in our genome, they continue to influence not only our genome architecture but also gene functioning in a broader sense.
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Background Enhancers are one of the most important classes of cis-regulatory elements (CREs) and play key roles in regulation of transcription in higher eukaryotes. Enhancers are difficult to identify because they lack positional constraints relative to their cognate genes. Excitingly, several recent studies showed that plant enhancers can be predicted based on their distinct features associated with open chromatin. However, experimental validation is necessary to confirm the predicted enhancer function. Results We developed a rapid enhancer validation system based on Nicotiana benthamiana. A set of 12 intergenic and intronic enhancers, identified in Arabidopsis thaliana, were cloned into a vector containing a minimal 35S promoter and a luciferase reporter gene, and were then infiltrated into N. benthamiana leaves mediated by agrobacterium. The enhancer activity of each candidate was quantitatively assayed based on bioluminescence measurement. The data from this luciferase-based validation was correlated with previous data derived from transgenic assays in A. thaliana. In addition, the relative strength of different enhancers for driving the reporter gene can be quantitatively compared. We demonstrate that this system can also be used to map the functional activity of a candidate enhancer under different environmental conditions. Conclusions In summary, we developed a rapid and efficient plant enhancer validation system based on a luciferase reporter and N. benthamiana-based leaf agroinfiltration. This system can be used for initial screening of leaf-specific enhancers and for validating candidate leaf enhancers from dicot species. It can potentially be used to examine the activity of candidate enhancers under different environmental conditions. Electronic supplementary material The online version of this article (10.1186/s13007-019-0407-y) contains supplementary material, which is available to authorized users.
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Background: Increased transcription of the human endogenous retrovirus group HERV-K (HML-2) is often seen during disease. Although the mechanism of its tissue-specific activation is unclear, research shows that LTR CpG hypomethylation alone is not sufficient to induce its promoter activity and that the transcriptional milieu of a malignant cell contributes, at least partly, to differential HML-2 expression. Results: We analyzed the relationship between LTR sequence variation and promoter expression patterns in human breast cancer cell lines, finding them to be positively correlated. In particular, two proviruses (3q12.3 and 11p15.4) displayed increased activity in almost all tumorigenic cell lines sampled. Using a transcription factor binding site prediction algorithm, we identified two unique binding sites in each 5' LTR that appeared to be associated with inducing promoter activity during neoplasia. Genomic analysis of the homologous proviruses in several non-human primates indicated post-integration genetic drift in two transcription factor binding sites, away from the ancestral sequence and towards the active form. Based on the sequences of 2504 individuals from the 1000 Genomes Project, the active form of the 11p15.4 site was found to be polymorphic within the human population, with an allele frequency of 51%, whereas the activating mutation in the 3q12.3 provirus was fixed in humans but not present in the orthologous provirus in chimpanzees or gorillas. Conclusions: These data suggest that stage-specific transcription factors at least partly contribute to LTR promoter activity during transformation and that, in some cases, transcription factor binding site polymorphisms may be responsible for the differential HML-2 expression often seen between individuals.
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We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ∼30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ∼3 Mb). We developed a protocol to generate ultra-long reads (N50 > 100 kb, read lengths up to 882 kb). Incorporating an additional 5× coverage of these ultra-long reads more than doubled the assembly contiguity (NG50 ∼6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length, and closure of gaps in the reference human genome assembly GRCh38.
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The imprinted gene PLAGL1 is an important regulator of apoptosis and cell cycle arrest. Loss of its expression has been implicated in tumorigenesis in a range of different cancers, and overexpression during fetal development causes transient neonatal diabetes mellitus (TNDM). PLAGL1 lies within an imprinted region of chromosome 6q24, and monoallelic expression from the major, differentially methylated promoter (P1) occurs in most human tissues. However, in peripheral blood leukocytes, the active promoter (P2) is non-imprinted and drives biallelic transcription. We report here a novel PLAGL1 promoter (P5) derived from the insertion of a primate-specific, MIR3 SINE retrotransposon. P5 is highly utilized in lymphocytes, particularly in T cells, and like P2, directs biallelic transcription. Our results show that it is important to consider P5 in relation to PLAGL1 function in T cells when investigating the dysregulation of this gene.
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The contribution of the transposons’ promoter in the horizontal transfer process is quite overlooked in the scientific literature. To shed light on this aspect we have mimicked the horizontal transfer process in laboratory and assayed in a wide range of hosts (fly, human, yeast and bacteria) the promoter activity of the 5′ terminal sequences in Bari1 and Bari3, two Drosophila transposons belonging to the Tc1-mariner superfamily. These sequences are able to drive the transcription of a reporter gene even in distantly related organisms at least at the episomal level. By combining bioinformatics and experimental approaches, we define two distinct promoter sequences for each terminal sequence analyzed, which allow transcriptional activity in prokaryotes and eukaryotes, respectively. We propose that the Bari family of transposons, and possibly other members of the Tc1-mariner superfamily, might have evolved “blurry promoters,” which have facilitated their diffusion in many living organisms through horizontal transfer.
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Transposable elements (TE), small mobile genetic elements unable to exist independently of the host genome, were initially believed to be exclusively deleterious genomic parasites. However, it is now clear that they play an important role as bacterial mutagenic agents, enabling the host to adapt to new environmental challenges and to colonize new niches. This review focuses on the impact of insertion sequences (IS), arguably the smallest TE, on bacterial genome plasticity and concomitant adaptability of phenotypic traits, including resistance to antibacterial agents, virulence, pathogenicity and catabolism. The direct consequence of IS transposition is the insertion of one DNA sequence into another. This event can result in gene inactivation as well as in modulation of neighbouring gene expression. The latter is usually mediated by de-repression or by the introduction of a complete or partial promoter located within the element. Furthermore, transcription and transposition of IS are affected by host factors and in some cases by environmental signals offering the host an adaptive strategy and promoting genetic variability to withstand the environmental challenges.
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Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical implementation requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB’s genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age-related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.
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Accurate computational identification of promoters remains a challenge as these key DNA regulatory regions have variable structures composed of functional motifs that provide gene specific initiation of transcription. In this paper we utilize Convolutional Neural Networks (CNN) to analyze sequence characteristics of prokaryotic and eukaryotic promoters and build their predictive models. We trained the same CNN architecture on promoters of four very distant organisms: human, plant (Arabidopsis), and two bacteria (Escherichia coli and Mycoplasma pneumonia). We found that CNN trained on sigma70 subclass of Escherichia coli promoter gives an excellent classification of promoters and non-promoter sequences (Sn=0.90, Sp=0.96, CC=0.84). The Bacillus subtilis promoters identification CNN model achieves Sn=0.91, Sp=0.95, and CC=0.86. For human and Arabidopsis promoters we employ CNNs for identification of two well-known promoter classes (TATA and non-TATA promoters). CNNs models nicely recognize these complex functional regions. For human Sn/Sp/CC accuracy of prediction reached 0.95/0.98/0,90 on TATA and 0.90/0.98/0.89 for non-TATA promoter sequences, respectively. For Arabidopsis we observed Sn/Sp/CC 0.95/0.97/0.91 (TATA) and 0.94/0.94/0.86 (non-TATA) promoters. Thus, the developed CNN models (implemented in CNNProm program) demonstrated the ability of deep learning with grasping complex promoter sequence characteristics and achieve significantly higher accuracy compared to the previously developed promoter prediction programs. As the suggested approach does not require knowledge of any specific promoter features, it can be easily extended to identify promoters and other complex functional regions in sequences of many other and especially newly sequenced genomes. The CNNProm program is available to run at web server http://www.softberry.com.
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The term heterochromatin has been long considered synonymous with gene silencing, but it is now clear that the presence of transcribed genes embedded in pericentromeric heterochromatin is a conserved feature in the evolution of eukaryotic genomes. Several studies have addressed the epigenetic changes that enable the expression of genes in pericentric heterochromatin, yet little is known about the evolutionary processes through which this has occurred. By combining genome annotation analysis and high-resolution cytology, we have identified and mapped 53 orthologs of D. melanogaster heterochromatic genes in the genomes of two evolutionarily distant species, D. pseudoobscura and D. virilis. Our results show that the orthologs of the D. melanogaster heterochromatic genes are clustered at three main genomic regions in D. virilis and D. pseudoobscura. In D. virilis, the clusters lie in the middle of euchromatin, while those in D. pseudoobscura are located in the proximal portion of the chromosome arms. Some orthologs map to the corresponding Muller C element in D. pseudoobscura and D. virilis, while others localize on the Muller B element, suggesting that chromosomal rearrangements that have been instrumental in the fusion of two separate elements involved the progenitors of genes currently located in D. melanogaster heterochromatin. These results demonstrate an evolutionary repositioning of gene clusters from ancestral locations in euchromatin to the pericentromeric heterochromatin of descendent D. melanogaster chromosomes. Remarkably, in both D. virilis and D. pseudoobscura the gene clusters show a conserved association with the HP1a protein, one of the most highly evolutionarily conserved epigenetic marks. In light of these results, we suggest a new scenario whereby ancestral HP1-like proteins (and possibly other epigenetic marks) may have contributed to the evolutionary repositioning of gene clusters into heterochromatin.
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Promoters initiate RNA synthesis, and enhancers stimulate promoter activity. Whether promoter and enhancer activities are encoded distinctly in DNA sequences is unknown. We measured the enhancer and promoter activities of thousands of DNA fragments transduced into mouse neurons. We focused on genomic loci bound by the neuronal activity-regulated co-activator CREBBP, and we measured enhancer and promoter activities both before and after neuronal activation. We find that the same sequences typically encode both enhancer and promoter activities. However, gene promoters generate more promoter activity than distal enhancers, despite generating similar enhancer activity. Surprisingly, the greater promoter activity of gene promoters is not due to conventional core promoter elements or splicing signals. Instead, we find that particular transcription factor binding motifs are intrinsically biased toward the generation of promoter activity, while others are not. While the specific biases we observe may be dependent on experimental or cellular context, our results suggest that gene promoters are distinguished from distal enhancers by specific complements of transcriptional activators.
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The term “Genetic modified organisms (GMO)” has become a controversial topic as its benefits for both food producers and consumers are companied by potential biomedical risks and environmental side effects. Increasing concerns from the public about GMO, particularly in the form of genetic modified (GM) foods, are aimed at the short- and long-lasting health problems that may result from this advanced biotechnology. Complex studies are being carried out around the world independently to evaluate the advantages and disadvantages of GM foods. In this paper, we attempt to summarize up-to-date knowledge about the benefits and potential problems of GM food. We also introduce some recent technological developments in GM foods and their impact in the field.
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The tetracycline-controlled Tet-Off and Tet-On gene expression systems are used to regulate the activity of genes in eukaryotic cells in diverse settings, varying from basic biological research to biotechnology and gene therapy applications. These systems are based on regulatory elements that control the activity of the tetracycline-resistance operon in bacteria. The Tet-Off system allows silencing of gene expression by administration of tetracycline (Tc) or tetracycline-derivatives like doxycycline (dox), whereas the Tet-On system allows activation of gene expression by dox. Since the initial design and construction of the original Tet-system, these bacterium-derived systems have been significantly improved for their function in eukaryotic cells. We here review how a dox-controlled HIV-1 variant was designed and used to greatly improve the activity and dox-sensitivity of the rtTA transcriptional activator component of the Tet-On system. These optimized rtTA variants require less dox for activation, which will reduce side effects and allow gene control in tissues where a relatively low dox level can be reached, such as the brain.
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Bari elements are members of the Tc1-mariner superfamily of DNA transposons, originally discovered in Drosophila melanogaster, and subsequently identified in silico in 11 sequenced Drosophila genomes and as experimentally isolated in four non-sequenced Drosophila species. Bari-like elements have been also studied for their mobility both in vivo and in vitro. We analyzed 23 Drosophila genomes and carried out a detailed characterization of the Bari elements identified, including those from the heterochromatic Bari1 cluster in D. melanogaster. We have annotated 401 copies of Bari elements classified either as putatively autonomous or inactive according to the structure of the terminal sequences and the presence of a complete transposase-coding region. Analyses of the integration sites revealed that Bari transposase prefers AT-rich sequences in which the TA target is cleaved and duplicated. Furthermore evaluation of transposon’s co-occurrence near the integration sites of Bari elements showed a non-random distribution of other transposable elements. We also unveil the existence of a putatively autonomous Bari1 variant characterized by two identical long Terminal Inverted Repeats, in D. rhopaloa. In addition, we detected MITEs related to Bari transposons in 9 species. Phylogenetic analyses based on transposase gene and the terminal sequences confirmed that Bari-like elements are distributed into three subfamilies. A few inconsistencies in Bari phylogenetic tree with respect to the Drosophila species tree could be explained by the occurrence of horizontal transfer events as also suggested by the results of dS analyses. This study further clarifies the Bari transposon’s evolutionary dynamics and increases our understanding on the Tc1-mariner elements’ biology.
Book
This open access book addresses the challenge of analyzing and understanding the evolutionary dynamics of complex biological systems at the genomic level, and elaborates on some promising strategies that would bring us closer to uncovering of the vital relationships between genotype and phenotype. After a few educational primers, the book continues with sections on sequence homology and alignment, phylogenetic methods to study genome evolution, methodologies for evaluating selective pressures on genomic sequences as well as genomic evolution in light of protein domain architecture and transposable elements, population genomics and other omics, and discussions of current bottlenecks in handling and analyzing genomic data. Written for the highly successful Methods in Molecular Biology series, chapters include the kind of detail and expert implementation advice that lead to the best results. Authoritative and comprehensive, Evolutionary Genomics: Statistical and Computational Methods, Second Edition aims to serve both novices in biology with strong statistics and computational skills, and molecular biologists with a good grasp of standard mathematical concepts, in moving this important field of study forward.
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Tools for tuning transcription in mammalian cells have broad applications, from basic biological discovery to human gene therapy. While precise control over target gene transcription via dosing with small molecules (drugs) is highly sought, the design of such inducible systems that meets required performance metrics poses a great challenge in mammalian cell synthetic biology. Important characteristics include tight and tunable gene expression with a low background, minimal drug toxicity, and orthogonality. Here, we review small-molecule-inducible transcriptional control devices that have demonstrated success in mammalian cells and mouse models. Most of these systems employ natural or designed ligand-binding protein domains to directly or indirectly communicate with transcription machinery at a target sequence, via carefully constructed fusions. Example fusions include those to transcription activator-like effectors (TALEs), DNA-targeting proteins (e.g. dCas systems) fused to transactivating domains, and recombinases. Similar to the architecture of Type I nuclear receptors, many of the systems are designed such that the transcriptional controller is excluded from the nucleus in the absence of an inducer. Techniques that use ligand-induced proteolysis and antibody-based chemically induced dimerizers are also described. Collectively, these transcriptional control devices take advantage of a variety of recently developed molecular biology tools and cell biology insights and represent both proof of concept (e.g. targeting reporter gene expression) and disease-targeting studies.
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Eukaryotic gene regulation is mediated by cis -regulatory elements, which are embedded within the vast non-coding genomic space and recognized by the transcription factors in a sequence- and context-dependent manner. A large proportion of eukaryotic genomes, including at least half of the human genome, are composed of transposable elements (TEs), which in their ancestral form carried their own cis -regulatory sequences able to exploit the host trans environment to promote TE transcription and facilitate transposition. Although not all present-day TE copies have retained this regulatory function, the preexisting regulatory potential of TEs can provide a rich source of cis -regulatory innovation for the host. Here, we review recent evidence documenting diverse contributions of TE sequences to gene regulation by functioning as enhancers, promoters, silencers and boundary elements. We discuss how TE-derived enhancer sequences can rapidly facilitate changes in existing gene regulatory networks and mediate species- and cell-type-specific regulatory innovations, and we postulate a unique contribution of TEs to species-specific gene expression divergence in pluripotency and early embryogenesis. With advances in genome-wide technologies and analyses, systematic investigation of TEs' cis -regulatory potential is now possible and our understanding of the biological impact of genomic TEs is increasing. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.
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The proper activities of enhancers and gene promoters are essential for coordinated transcription within a cell. Although diverse methodologies have been developed to identify enhancers and promoters, most have tacitly assumed that these elements are distinct. However, studies have unexpectedly shown that regulatory elements may have both enhancer and promoter functions. Here we review these results, focusing on the factors that determine the promoter and/or enhancer activity of regulatory elements. We discuss emerging models that define regulatory elements by accessible DNA and their non-mutually-exclusive abilities to drive transcription initiation (promoter activity) and/or to enhance transcription at other such regions (enhancer activity).
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Constitutive heterochromatin represents a significant portion of eukaryotic genomes, but its functions still need to be elucidated. Even in the most updated genetics and molecular biology textbooks, constitutive heterochromatin is portrayed mainly as the 'silent' component of eukaryotic genomes. However, there may be more complexity to the relationship between heterochromatin and gene expression. In the fruit fly Drosophila melanogaster, a model for heterochromatin studies, about one-third of the genome is heterochromatic and is concentrated in the centric, pericentric, and telomeric regions of the chromosomes. Recent findings indicate that hundreds of D. melanogaster genes can 'live and work' properly within constitutive heterochromatin. The genomic size of these genes is generally larger than that of euchromatic genes and together they account for a significant fraction of the entire constitutive heterochromatin. Thus, this peculiar genome component in spite its ability to induce silencing, has in fact the means for being quite dynamic. A major scope of this review is to revisit the 'dogma of silent heterochromatin'.
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The use of GM animals to produce human recombinant therapeutic proteins has progressed only slowly. But recent successes and new technologies could speed up development.
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During embryogenesis, precise gene transcription in space and time requires that distal enhancers and promoters communicate by physical proximity within gene regulatory landscapes. To achieve this, regulatory landscapes fold in nuclear space, creating complex 3D structures that influence enhancer-promoter communication and gene expression and that, when disrupted, can cause disease. Here, we provide an overview of how enhancers and promoters construct regulatory landscapes and how multiple scales of 3D chromatin structure sculpt their communication. We focus on emerging views of what enhancer-promoter contacts and chromatin domains physically represent and how two antagonistic fundamental forces-loop extrusion and homotypic attraction-likely form them. We also examine how these same forces spatially separate regulatory landscapes by functional state, thereby creating higher-order compartments that reconfigure during development to enable proper enhancer-promoter communication.
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Despite the importance of Y‐chromosomes in evolution and sex determination, their heterochromatic, repeat‐rich nature makes them difficult to sequence (due, in part, to ambiguities in sequence alignment and assembly) and to genetically manipulate, and therefore they generally remain poorly understood. For example, the D. melanogaster Y‐chromosome, one of the most extensively studied Y‐chromosomes, is widely heterochromatic and composed mainly of highly repetitive sequences, with only a handful of expressed genes scattered throughout its length. Efforts to insert transgenes on this chromosome have thus far relied on either random insertion of transposons (sometimes harboring ‘landing sites’ for subsequent integrations) with limited success or on chromosomal translocations, thereby limiting the types of Y‐chromosome related questions that could be explored. Here we describe a versatile approach to site‐specifically insert transgenes on the Y‐chromosome in D. melanogaster via CRISPR/Cas9‐mediated Homology Directed Repair (HDR). We demonstrate the ability to insert, and detect expression from, fluorescently marked transgenes at two specific locations on the Y‐chromosome, and we utilize these marked Y‐chromosomes to detect and quantify rare chromosomal nondisjunction effects. Finally, we discuss how this Y‐docking technique could be adapted to other insects to aid in the development of genetic control technologies for the management of insect disease vectors and pests. This article is protected by copyright. All rights reserved.
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We applied a combinatorial indexing assay, sci-ATAC-seq, to profile genome-wide chromatin accessibility in ∼100,000 single cells from 13 adult mouse tissues. We identify 85 distinct patterns of chromatin accessibility, most of which can be assigned to cell types, and ∼400,000 differentially accessible elements. We use these data to link regulatory elements to their target genes, to define the transcription factor grammar specifying each cell type, and to discover in vivo correlates of heterogeneity in accessibility within cell types. We develop a technique for mapping single cell gene expression data to single-cell chromatin accessibility data, facilitating the comparison of atlases. By intersecting mouse chromatin accessibility with human genome-wide association summary statistics, we identify cell-type-specific enrichments of the heritability signal for hundreds of complex traits. These data define the in vivo landscape of the regulatory genome for common mammalian cell types at single-cell resolution.
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RNA polymerase II (Pol II) core promoters are specialized DNA sequences at transcription start sites of protein-coding and non-coding genes that support the assembly of the transcription machinery and transcription initiation. They enable the highly regulated transcription of genes by selectively integrating regulatory cues from distal enhancers and their associated regulatory proteins. In this Review, we discuss the defining properties of gene core promoters, including their sequence features, chromatin architecture and transcription initiation patterns. We provide an overview of molecular mechanisms underlying the function and regulation of core promoters and their emerging functional diversity, which defines distinct transcription programmes. On the basis of the established properties of gene core promoters, we discuss transcription start sites within enhancers and integrate recent results obtained from dedicated functional assays to propose a functional model of transcription initiation. This model can explain the nature and function of transcription initiation at gene starts and at enhancers and can explain the different roles of core promoters, of Pol II and its associated factors and of the activating cues provided by enhancers and the transcription factors and cofactors they recruit.
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Retrotransposons encompass half of the human genome and contribute to the formation of heterochromatin, which provides nuclear structure and regulates gene expression. Here, we asked if the human silencing hub (HUSH) complex is necessary to silence retrotransposons and whether it collaborates with TRIM28 and the chromatin remodeler ATRX at specific genomic loci. We show that the HUSH complex contributes to de novo repression and DNA methylation of a SVA retrotransposon reporter. By using naïve vs. primed mouse pluripotent stem cells, we reveal a critical role for the HUSH complex in naïve cells, implicating it in programming epigenetic marks in development. While the HUSH component FAM208A binds to endogenous retroviruses (ERVs) and long interspersed element-1s (LINE-1s or L1s), it is mainly required to repress evolutionarily young L1s (mouse-specific lineages less than 5 million years old). TRIM28, in contrast, is necessary to repress both ERVs and young L1s. Genes co-repressed by TRIM28 and FAM208A are evolutionarily young, or exhibit tissue-specific expression, are enriched in young L1s and display evidence for regulation through LTR promoters. Finally, we demonstrate that the HUSH complex is also required to repress L1 elements in human cells. Overall, these data indicate that the HUSH complex and TRIM28 co-repress young retrotransposons and new genes rewired by retrotransposon non-coding DNA.
Chapter
This chapter deals with natural and synthetic promoters frequently used in yeast. It describes principles and strategies exploited to produce synthetic promoters and their cognate transcription factors. The chapter points out the essential structural and functional features of yeast promoters. It highlights some examples of both regulated and constitutive natural yeast promoters. The characterization of these sequences allowed for the identification of structural and functional features that are exploited to build synthetic promoters and heterologous transcription factors. Modification of the binding affinity between the transcription factor and its cognate transcription factor binding site (TFBS) results in promoter strength modulation. The chapter discusses two main groups of synthetic promoters. One includes modified versions of natural promoters, and the other contains hybrid promoters. The chapter also describes Saccharomyces cerevisiae promoters. The characterization of S. cerevisiae promoters began by using them to drive expression of reporter genes.
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It is becoming clear that most eukaryotic transposable elements (TEs) owe their evolutionary success in part to horizontal transfer events, which enable them to invade new species. Recent large-scale studies are beginning to unravel the mechanisms and ecological factors underlying this mode of transmission. Viruses are increasingly recognized as vectors in the process but also as a direct source of genetic material horizontally acquired by eukaryotic organisms. Because TEs and endogenous viruses are major catalysts of variation and innovation in genomes, we argue that horizontal inheritance has had a more profound impact in eukaryotic evolution than is commonly appreciated. To support this proposal, we compile a list of examples, including some previously unrecognized, whereby new host functions and phenotypes can be directly attributed to horizontally acquired TE or viral sequences. We predict that the number of examples will rapidly grow in the future as the prevalence of horizontal transfer in the life cycle of TEs becomes even more apparent, firmly establishing this form of non-Mendelian inheritance as a consequential facet of eukaryotic evolution.
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This review presents some of the hottest topics in biotechnological applications: proteases in biocatalysis. Obviously, one of the most relevant areas of application is in the hydrolysis of proteins in food technology, and that has led to a massive use on proteomics. The aim is to identify via peptide maps the different proteins obtained after a specific protease hydrolysis. However, concepts like degradomics are also taking on a more relevant importance in the use and study of proteases and will also be discussed. Other protease applications, as seem in cleaning (detergent development), the pharmaceutical industry, and in fine chemistry, will be analyzed. This review progresses from basic areas such as protease classification to a discussion of the preparation of protease-immobilized biocatalysts, considering the different problems raised by the use of immobilized proteases due to the peculiar features of the substrates, usually large macromolecules. Production of bioactive peptides via limited hydrolysis of proteins will occupy an important place in this review.
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Evolution of cis-properties (such as enhancers) often plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods to study enhancers in species outside of established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, Tribolium castaneum. To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of Tribolium via FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and stage. Second, we established the first reporter assay system that works in both Drosophila and Tribolium, using nubbin in the wing and hunchback in the embryo as case studies. Together, these advances will be useful to study the evolution of cis-language and morphological diversity in Tribolium and other insects.
Chapter
Transposition plays a major role in genome plasticity causing chromosome rearrangements, deoxyribonucleic acid (DNA) insertion and deletion mutations and is key in gene sequestration and transmission by horizontal gene transfer. The processes play important roles in genome evolution and function. Transposable elements (TR) can be classified according to the chemistry of the transposition process itself: the nature of the catalytic mechanisms involved in the DNA cleavages necessary to liberate a copy of the transposon from its donor DNA and DNA strand transfer events (involved in insertion into a target DNA). This chapter is centred on prokaryotic TE, which use DNA intermediates for their movement. DNA transposable elements are extremely diverse, but, with a few exceptions, the mechanisms they use in their movement are common to both prokaryotic and eukaryotic TE. There are only a limited number of enzyme types that carry out DNA transposition. They are DDE (DED), Y, S, Y1 (HUH), Y2 (HUH) transposases, names of which are based on the active-site residues involved in catalysis. More recently, an additional class, the casposons, has been identified in archaea and bacteria, which use a different type of sequence-specific endonuclease. Transposition requires assembly of precise protein DNA complexes called transpososomes. Transposons are found in all living organisms.Transposons are diverse in organisation.Transposons use a limited number of enzyme types (transposases) for their movement.Transposases catalyse DNA cleavage in the donor DNA molecule and DNA strand transfer into the target DNA.Transposition requires an assembly of a precise protein–DNA complex, the transpososome.Transpososomes are assembled through a strictly ordered series of events in which the DNA–protein interactions position the DNA for catalysis.
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Promoter functionality is highly context dependent, as exemplified by gene-specific expression profiles across different tissues and cell types. Cell type-specific promoter regulation is a function of each cell’s unique complement of transcriptional machinery components. Accordingly, to achieve high levels of transcriptional activity within a particular cell type, synthetic promoters must be specifically designed to harness those cells discrete repertoire of available transcription factors. Here, we describe a method for constructing very strong cell type-specific synthetic promoters for use in any given mammalian host cell. Transcription factor regulatory elements (TFREs; or transcription factor binding sites) that can independently mediate activation of recombinant gene transcription in the chosen host cells by using available transcription factor activity are identified and utilized as building blocks to construct novel promoter sequences with varying activities. Bioinformatics analysis of synthetic promoter ’s TFRE compositions is then performed to determine how differing relative TFRE abundances explain variations in relative promoter activities. This information is used to derive an optimal second-generation promoter library construction design space, such that promoters with maximal transcriptional activity in the host cell type can be created.
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Transposable elements (TEs) are a prolific source of tightly regulated, biochemically active non-coding elements, such as transcription factor-binding sites and non-coding RNAs. Many recent studies reinvigorate the idea that these elements are pervasively co-opted for the regulation of host genes. We argue that the inherent genetic properties of TEs and the conflicting relationships with their hosts facilitate their recruitment for regulatory functions in diverse genomes. We review recent findings supporting the long-standing hypothesis that the waves of TE invasions endured by organisms for eons have catalysed the evolution of gene-regulatory networks. We also discuss the challenges of dissecting and interpreting the phenotypic effect of regulatory activities encoded by TEs in health and disease.
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Gene expression in bacteria relies on promoter recognition by the DNA-dependent RNA polymerase and subsequent transcription initiation. Bacterial cells are able to tune their transcriptional programmes to changing environments, through numerous mechanisms that regulate the activity of RNA polymerase, or change the set of promoters to which the RNA polymerase can bind. In this Review, we outline our current understanding of the different factors that direct the regulation of transcription initiation in bacteria, whether by interacting with promoters, with RNA polymerase or with both, and we discuss the diverse molecular mechanisms that are used by these factors to regulate gene expression.