PIWI-interacting RNAs (piRNAs) play a crucial role in safeguarding genome integrity by silencing mobile genetic elements. From flies to humans, piRNAs originate from long single-stranded precursors encoded by genomic piRNA clusters. How piRNA clusters form to adapt to genomic invaders and evolve to maintain protection remain key outstanding questions. Here, we generate a roadmap of piRNA clusters across seven species that highlights both similarities and variations. In mammals, we identify transcriptional readthrough as a mechanism to generate piRNAs from transposon insertions (piCs) downstream of genes (DoG). Together with the well-known stress-dependent DoG transcripts, our findings suggest a molecular mechanism for the formation of piRNA clusters in response to retroviral invasion. Finally, we identify a class of dynamic piRNA clusters in humans, underscoring unique features of human germ cell biology. Our results advance the understanding of conserved principles and species-specific variations in piRNA biology and provide tools for future studies.
- MeSH
- druhová specificita MeSH
- lidé MeSH
- malá interferující RNA * metabolismus genetika MeSH
- myši MeSH
- Piwi-interagující RNA MeSH
- psi MeSH
- savci * genetika MeSH
- transpozibilní elementy DNA genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The recent revision of the Acidithiobacillia class using genomic taxonomy methods has shown that, in addition to the existence of previously unrecognized genera and species, some species of the class harbor levels of divergence that are congruent with ongoing differentiation processes. In this study, we have performed a subspecies-level analysis of sequenced strains of Acidithiobacillus ferrooxidans to prove the existence of distinct sublineages and identify the discriminant genomic/genetic characteristics linked to these sublineages, and to shed light on the processes driving such differentiation. Differences in the genomic relatedness metrics, levels of synteny, gene content, and both integrated and episomal mobile genetic elements (MGE) repertoires support the existence of two subspecies-level taxa within A. ferrooxidans. While sublineage 2A harbors a small plasmid related to pTF5, this episomal MGE is absent in sublineage 2B strains. Likewise, clear differences in the occurrence, coverage and conservation of integrated MGEs are apparent between sublineages. Differential MGE-associated gene cargo pertained to the functional categories of energy metabolism, ion transport, cell surface modification, and defense mechanisms. Inferred functional differences have the potential to impact long-term adaptive processes and may underpin the basis of the subspecies-level differentiation uncovered within A. ferrooxidans. Genome resequencing of iron- and sulfur-adapted cultures of a selected 2A sublineage strain (CCM 4253) showed that both episomal and large integrated MGEs are conserved over twenty generations in either growth condition. In turn, active insertion sequences profoundly impact short-term adaptive processes. The ISAfe1 element was found to be highly active in sublineage 2A strain CCM 4253. Phenotypic mutations caused by the transposition of ISAfe1 into the pstC2 encoding phosphate-transport system permease protein were detected in sulfur-adapted cultures and shown to impair growth on ferrous iron upon the switch of electron donor. The phenotypic manifestation of the △pstC2 mutation, such as a loss of the ability to oxidize ferrous iron, is likely related to the inability of the mutant to secure the phosphorous availability for electron transport-linked phosphorylation coupled to iron oxidation. Depletion of the transpositional △pstC2 mutation occurred concomitantly with a shortening of the iron-oxidation lag phase at later transfers on a ferrous iron-containing medium. Therefore, the pstII operon appears to play an essential role in A. ferrooxidans when cells oxidize ferrous iron. Results highlight the influence of insertion sequences and both integrated and episomal mobile genetic elements in the short- and long-term adaptive processes of A. ferrooxidans strains under changing growth conditions.
BACKGROUND: The insertion sequence elements (IS elements) represent the smallest and the most abundant mobile elements in prokaryotic genomes. It has been shown that they play a significant role in genome organization and evolution. To better understand their function in the host genome, it is desirable to have an effective detection and annotation tool. This need becomes even more crucial when considering rapid-growing genomic and metagenomic data. The existing tools for IS elements detection and annotation are usually based on comparing sequence similarity with a database of known IS families. Thus, they have limited ability to discover distant and putative novel IS elements. RESULTS: In this paper, we present digIS, a software tool based on profile hidden Markov models assembled from catalytic domains of transposases. It shows a very good performance in detecting known IS elements when tested on datasets with manually curated annotation. The main contribution of digIS is in its ability to detect distant and putative novel IS elements while maintaining a moderate level of false positives. In this category it outperforms existing tools, especially when tested on large datasets of archaeal and bacterial genomes. CONCLUSION: We provide digIS, a software tool using a novel approach based on manually curated profile hidden Markov models, which is able to detect distant and putative novel IS elements. Although digIS can find known IS elements as well, we expect it to be used primarily by scientists interested in finding novel IS elements. The tool is available at https://github.com/janka2012/digIS.
- MeSH
- genom bakteriální genetika MeSH
- genomika MeSH
- lidé MeSH
- prokaryotické buňky * MeSH
- software MeSH
- transpozibilní elementy DNA * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
DNA transposons are defined as repeated DNA sequences that can move within the host genome through the action of transposases. The transposon superfamily Merlin was originally found mainly in animal genomes. Here, we describe a global distribution of the Merlin in animals, fungi, plants and protists, reporting for the first time their presence in Rhodophyceae, Metamonada, Discoba and Alveolata. We identified a great variety of potentially active Merlin families, some containing highly imperfect terminal inverted repeats and internal tandem repeats. Merlin-related sequences with no evidence of mobilization capacity were also observed and may be products of domestication. The evolutionary trees support that Merlin is likely an ancient superfamily, with early events of diversification and secondary losses, although repeated re-invasions probably occurred in some groups, which would explain its diversity and discontinuous distribution. We cannot rule out the possibility that the Merlin superfamily is the product of multiple horizontal transfers of related prokaryotic insertion sequences. Moreover, this is the first account of a DNA transposon in kinetoplastid flagellates, with conserved Merlin transposase identified in Bodo saltans and Perkinsela sp., whereas it is absent in trypanosomatids. Based on the level of conservation of the transposase and overlaps of putative open reading frames with Merlin, we propose that in protists it may serve as a raw material for gene emergence.
Chimeric antigen receptor T-cells (CAR T-cells) represent a novel and promising approach in cancer immunotherapy. According to the World Health Organization (WHO), the number of oncological patients is steadily growing in developed countries despite immense progress in oncological treatments, and the prognosis of individual patients is still relatively poor. Exceptional results have been recorded for CAR T-cell therapy in patients suffering from B-cell malignancies. This success opens up the possibility of using the same approach for other types of cancers. To date, the most common method for CAR T-cell generation is the use of viral vectors. However, dealing with virus-derived vectors brings possible obstacles in the CAR T-cell manufacturing process owing to strict regulations and high cost demands. Alternative approaches may facilitate further development and the transfer of the method to clinical practice. The most promising substitutes for virus-derived vectors are transposon-derived vectors, most commonly sleeping beauty, which offer great coding capability and a safe integration profile while maintaining a relatively low production cost. This review is aimed at summarizing the state of the art of nonviral approaches in CAR T-cell generation, with a unique perspective on the conditions in clinical applications and current Good Manufacturing Practice. If CAR T-cell therapy is to be routinely used in medical practice, the manufacturing cost and complexity need to be as low as possible, and transposon-based vectors seem to meet these criteria better than viral-based vectors.
- MeSH
- buněčné kultury metody MeSH
- chimerické antigenní receptory genetika imunologie MeSH
- genetické vektory genetika MeSH
- imunoterapie adoptivní metody MeSH
- lidé MeSH
- nádory imunologie terapie MeSH
- T-lymfocyty imunologie transplantace MeSH
- technika přenosu genů * MeSH
- transpozibilní elementy DNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Stalk lodging, which is generally determined by stalk strength, results in considerable yield loss and has become a primary threat to maize (Zea mays) yield under high-density planting. However, the molecular genetic basis of maize stalk strength remains unclear, and improvement methods remain inefficient. Here, we combined map-based cloning and association mapping and identified the gene stiff1 underlying a major quantitative trait locus for stalk strength in maize. A 27.2-kb transposable element insertion was present in the promoter of the stiff1 gene, which encodes an F-box domain protein. This transposable element insertion repressed the transcription of stiff1, leading to the increased cellulose and lignin contents in the cell wall and consequently greater stalk strength. Furthermore, a precisely edited allele of stiff1 generated through the CRISPR/Cas9 system resulted in plants with a stronger stalk than the unedited control. Nucleotide diversity analysis revealed that the promoter of stiff1 was under strong selection in the maize stiff-stalk group. Our cloning of stiff1 reveals a case in which a transposable element played an important role in maize improvement. The identification of stiff1 and our edited stiff1 allele pave the way for efficient improvement of maize stalk strength.
- MeSH
- alely MeSH
- buněčná stěna metabolismus MeSH
- CRISPR-Cas systémy MeSH
- fenotyp MeSH
- kukuřice setá genetika MeSH
- lignin metabolismus MeSH
- lokus kvantitativního znaku MeSH
- mapování chromozomů MeSH
- promotorové oblasti (genetika) * MeSH
- rostlinné geny MeSH
- rostlinné proteiny genetika metabolismus MeSH
- sekvenční analýza MeSH
- transformace genetická MeSH
- transpozibilní elementy DNA genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The efficiency of chimeric antigen receptor (CAR) T-cell-based therapies depends on a sufficient expansion of CAR T cells in vivo and can be weakened by intra-tumoral suppression of CAR T cell functions, leading to a failure of therapy. For example, certain B-cell malignancies such as chronic lymphocytic leukemia are weakly sensitive to treatment with CAR T cells. Co-expression of proinflamatory cytokines such as IL-12 and IL-18 by CAR T cells have been shown to enhance their antitumor function. We similarly engineered CAR T cell to co-express IL-21 and studied the effects of IL-21 on CAR T cells specific to CD19 and prostate-specific membrane antigens using an in vitro co-culture model and NSG mice transplanted with B-cell tumors. RESULTS: IL-21 enhanced the expansion of CAR T cells after antigenic stimulation, reduced the level of apoptosis of CAR T cells during co-culture with tumor cells and prevented differentiation of CAR T cells toward late memory phenotypes. In addition, induced secretion of IL-21 by CAR T cells promoted tumor infiltration by CD19-specific CAR (CAR19) T cells in NSG mice, resulting in reduced tumor growth. By co-culturing CAR19 T cells with bone-marrow fragments infiltrated with CLL cells we demonstrate that IL-21 reduces the immunosupressive activity of CLL cells against CAR19 T cells. CONCLUSIONS: CAR19 T cells armed with IL-21 exhibited enhanced antitumor functions. IL-21 promoted their proliferation and cytotoxicity against chronic lymphocytic leukemia (CLL). The results suggest that arming CAR T cells with IL-21 could boost the effectiveness of CAR T-mediated therapies.
- MeSH
- fenotyp MeSH
- imunosupresivní léčba MeSH
- interleukiny metabolismus MeSH
- lidé MeSH
- myši MeSH
- nádory imunologie terapie MeSH
- proliferace buněk MeSH
- receptory antigenů T-buněk metabolismus MeSH
- T-lymfocyty imunologie MeSH
- transpozibilní elementy DNA genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The genomic shock hypothesis suggests that allopolyploidy is associated with genome changes driven by transposable elements, as a response to imbalances between parental insertion loads. To explore this hypothesis, we compared three allotetraploids, Nicotiana arentsii, N. rustica and N. tabacum, which arose over comparable time frames from hybridisation between increasingly divergent diploid species. We used sequence-specific amplification polymorphism (SSAP) to compare the dynamics of six transposable elements in these allopolyploids, their diploid progenitors and in corresponding synthetic hybrids. We show that element-specific dynamics in young Nicotiana allopolyploids reflect their dynamics in diploid progenitors. Transposable element mobilisation is not concomitant with immediate genome merger, but occurs within the first generations of allopolyploid formation. In natural allopolyploids, such mobilisations correlate with imbalances in the repeat profile of the parental species, which increases with their genetic divergence. Other restructuring leading to locus loss is immediate, nonrandom and targeted at specific subgenomes, independently of cross orientation. The correlation between transposable element mobilisation in allopolyploids and quantitative imbalances in parental transposable element loads supports the genome shock hypothesis proposed by McClintock.
We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, blaKPC-2 was harbored on a novel variant of a Tn4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, blaKPC-3 was on a Tn4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion. The interplay among plasmids carrying blaKPC and the coresident IncA plasmids offers new information on plasmids coresident within clinically relevant enterobacteria.
B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.
- MeSH
- B-buněčný lymfom genetika patologie MeSH
- buněčné klony MeSH
- CRISPR-Cas systémy genetika MeSH
- genetické asociační studie MeSH
- genetické testování metody MeSH
- genová dávka MeSH
- geny nádorové MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši transgenní MeSH
- receptory antigenů B-buněk metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- reprodukovatelnost výsledků MeSH
- transpozibilní elementy DNA genetika MeSH
- tumor supresorové geny MeSH
- ztráta heterozygozity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
