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1.
DNA Res ; 31(2)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38447059

RESUMO

Transposable elements (TEs) mobility is capable of generating a large number of structural variants (SVs), which can have considerable potential as molecular markers for genetic analysis and molecular breeding in livestock. Our results showed that the pig genome contains mainly TE-SVs generated by short interspersed nuclear elements (51,873/76.49%), followed by long interspersed nuclear elements (11,131/16.41%), and more than 84% of the common TE-SVs (Minor allele frequency, MAF > 0.10) were validated to be polymorphic. Subsequently, we utilized the identified TE-SVs to gain insights into the population structure, resulting in clear differentiation among the three pig groups and facilitating the identification of relationships within Chinese local pig breeds. In addition, we investigated the frequencies of TEs in the gene coding regions of different pig groups and annotated the respective TE types, related genes, and functional pathways. Through genome-wide comparisons of Large White pigs and Chinese local pigs utilizing the Beijing Black pigs, we identified TE-mediated SVs associated with quantitative trait loci and observed that they were mainly involved in carcass traits and meat quality traits. Lastly, we present the first documented evidence of TE transduction in the pig genome.


Assuntos
Elementos de DNA Transponíveis , Polimorfismo Genético , Animais , Suínos/genética , Locos de Características Quantitativas , Elementos Nucleotídeos Curtos e Dispersos , Genética Populacional
2.
Proc Natl Acad Sci U S A ; 121(11): e2309263121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38457521

RESUMO

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.


Assuntos
Conjugação Genética , Transferência Genética Horizontal , Filogenia , Transferência Genética Horizontal/genética , Evolução Biológica , Elementos de DNA Transponíveis/genética
3.
ACS Synth Biol ; 13(3): 901-912, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38445989

RESUMO

In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.


Assuntos
Anabaena , Cianobactérias , Humanos , RNA Guia de Sistemas CRISPR-Cas , RNA , Plasmídeos/genética , Anabaena/genética , Cianobactérias/genética , DNA , Escherichia coli/genética , Elementos de DNA Transponíveis/genética
4.
Planta ; 259(5): 99, 2024 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-38522063

RESUMO

MAIN CONCLUSION: Six grape centromere-specific markers for cytogenetics were mined by combining genetic and immunological assays, and the possible evolution mechanism of centromeric repeats was analyzed. Centromeric histone proteins are functionally conserved; however, centromeric repetitive DNA sequences may represent considerable diversity in related species. Therefore, studying the characteristics and structure of grape centromere repeat sequences contributes to a deeper understanding of the evolutionary process of grape plants, including their origin and mechanisms of polyploidization. Plant centromeric regions are mainly composed of repetitive sequences, including SatDNA and transposable elements (TE). In this research, the characterization of centromere sequences in the whole genome of grapevine (Vitis vinifera L.) has been conducted. Five centromeric tandem repeat sequences (Vv1, Vv2, Vv5, Vv6, and Vv8) and one long terminal repeat (LTR) sequence Vv24 were isolated. These sequences had different centromeric distributions, which indicates that grape centromeric sequences may undergo rapid evolution. The existence of extrachromosomal circular DNA (eccDNA) and gene expression in CenH3 subdomain region may provide various potential mechanisms for the generation of new centromeric regions.


Assuntos
Vitis , Vitis/genética , Centrômero/genética , Citoplasma , Elementos de DNA Transponíveis/genética , Histonas
5.
Proc Natl Acad Sci U S A ; 121(13): e2317095121, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38502704

RESUMO

To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified.


Assuntos
Heterocromatina , RNA de Interação com Piwi , Animais , Masculino , Camundongos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Metilação de DNA , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
6.
Sci Rep ; 14(1): 6756, 2024 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-38514891

RESUMO

Transposon directed insertion-site sequencing (TraDIS), a variant of transposon insertion sequencing commonly known as Tn-Seq, is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often requires laborious, time-consuming modifications to its protocol. In this technical study, we aimed to refine the protocol by identifying key parameters that can impact the complexity of mutant libraries. Firstly, we discovered that adjusting electroporation parameters including transposome concentration, transposome assembly conditions, and cell densities can significantly improve the recovery of viable mutants for different Escherichia coli strains. Secondly, we found that post-electroporation conditions, such as recovery time and the use of different mediums for selecting mutants may also impact the complexity of viable mutants in the library. Finally, we developed a simplified sequencing library preparation workflow based on a Nextera-TruSeq hybrid design where ~ 80% of sequenced reads correspond to transposon-DNA junctions. The technical improvements presented in our study aim to streamline TraDIS protocols, making this powerful technique more accessible for a wider scientific audience.


Assuntos
Elementos de DNA Transponíveis , Genes Bacterianos , Mutagênese Insercional , Elementos de DNA Transponíveis/genética , Análise Custo-Benefício , Sequência de Bases , Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biblioteca Gênica
8.
Sci Transl Med ; 16(738): eadj9283, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38478628

RESUMO

Genetic changes in repetitive sequences are a hallmark of cancer and other diseases, but characterizing these has been challenging using standard sequencing approaches. We developed a de novo kmer finding approach, called ARTEMIS (Analysis of RepeaT EleMents in dISease), to identify repeat elements from whole-genome sequencing. Using this method, we analyzed 1.2 billion kmers in 2837 tissue and plasma samples from 1975 patients, including those with lung, breast, colorectal, ovarian, liver, gastric, head and neck, bladder, cervical, thyroid, or prostate cancer. We identified tumor-specific changes in these patients in 1280 repeat element types from the LINE, SINE, LTR, transposable element, and human satellite families. These included changes to known repeats and 820 elements that were not previously known to be altered in human cancer. Repeat elements were enriched in regions of driver genes, and their representation was altered by structural changes and epigenetic states. Machine learning analyses of genome-wide repeat landscapes and fragmentation profiles in cfDNA detected patients with early-stage lung or liver cancer in cross-validated and externally validated cohorts. In addition, these repeat landscapes could be used to noninvasively identify the tissue of origin of tumors. These analyses reveal widespread changes in repeat landscapes of human cancers and provide an approach for their detection and characterization that could benefit early detection and disease monitoring of patients with cancer.


Assuntos
Ácidos Nucleicos Livres , Neoplasias Hepáticas , Masculino , Humanos , Neoplasias Hepáticas/genética , Elementos de DNA Transponíveis
9.
Int J Food Microbiol ; 414: 110629, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38368793

RESUMO

The rise of antibiotic resistance in Escherichia coli has become a major global public health concern. While there is extensive research on antibiotic-resistant E. coli from human and animal sources, studies on vegetables and their environments are limited. This study investigated the prevalence and characteristics of ciprofloxacin-resistant (CIPR) E. coli in 13 types of edible raw vegetables, along with their irrigation water and soil in Shaanxi, China. Of 349 samples collected (157 vegetables, 59 water, and 133 soil), a total of 48 positive samples were detected, with one CIPRE. coli strain isolated from each sample being selected for further analyses. A striking observation was its high prevalence in irrigation water at 44.1 %, markedly exceeding that in vegetables (12.0 %) and soil (4.5 %). The susceptibility of Forty-eight CIPRE. coli isolates was evaluated using the disc diffusion method for 18 different antibiotics, all these isolates were not only resistant to the tested fluoroquinolones antibiotics (levofloxacin, nalidixic acid), but also displayed a multi-drug resistance (MDR) pattern. Twenty-eight (58.3 %) of 48 CIPRE. coli isolates exhibited extended spectrum ß-lactamases (ESBLs) (CIPR-ESBLs) producing phenotype. Subsequently, whole-genome sequencing was performed on these 28 isolates. We identified 12 serotypes and STs each, with O101: H9 (35.7 %, 10/28) and ST10 (21.4 %, 6/28) being the most common. Further classification placed these isolates into five phylogenetic groups: A (57.1 %, 16/28), B1 (32.1 %, 9/28), D (3.6 %, 1/28), B2 (3.6 %,1/28), and F (3.6 %,1/28). Notelly, Identical ST types, serotypes and phylogroups were found in certain CIPR-ESBLs-producing E. coli from both vegetables and adjacent irrigation water. Genomic analysis of the 28 CIPR-ESBLs-producing E. coli isolates unveiled 73 resistance genes, associated with 13 amino acid mutations in resistance-determining regions (QRDRs) and resistance to 12 types of antibiotics. Each isolate was confirmed to carry both ESBLs and fluoroquinolone resistance genes, with the Ser83Ala mutation in GyrA (96.4 %, 27/28) being the most prevalent. A detailed analysis of Mobile Genetic Elements (MGEs) revealed that IncFIB and IncFII plasmid subtypes were most prevalent in 60.7 % and 67.9 % of isolates, respectively, with 75 % containing over 10 insertion sequences (IS) each. Furthermore, we observed that certain ESBL and PMQR genes were located on plasmids or in proximity to insertion sequences. In conclusion, our research highlights the widespread presence of CIPRE. coli in irrigation water and thoroughly examines the genetic characteristics of CIPR-ESBLs-producing E. coli strains, underlining the need for ongoing monitoring and management to reduce multidrug-resistant bacteria in vegetables and their environment.


Assuntos
Ciprofloxacina , Infecções por Escherichia coli , Animais , Humanos , Ciprofloxacina/farmacologia , Escherichia coli , Verduras/microbiologia , Elementos de DNA Transponíveis , Filogenia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Infecções por Escherichia coli/microbiologia , Fluoroquinolonas , Genômica , Água/metabolismo
10.
Nature ; 626(8001): 1116-1124, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38355802

RESUMO

Transposable elements (TEs) are a major constituent of human genes, occupying approximately half of the intronic space. During pre-messenger RNA synthesis, intronic TEs are transcribed along with their host genes but rarely contribute to the final mRNA product because they are spliced out together with the intron and rapidly degraded. Paradoxically, TEs are an abundant source of RNA-processing signals through which they can create new introns1, and also functional2 or non-functional chimeric transcripts3. The rarity of these events implies the existence of a resilient splicing code that is able to suppress TE exonization without compromising host pre-mRNA processing. Here we show that SAFB proteins protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity, via prevention of the exonization of previously integrated TEs. This unique dual role is possible because of L1's conserved adenosine-rich coding sequences that are bound by SAFB proteins. The suppressive activity of SAFB extends to tissue-specific, giant protein-coding cassette exons, nested genes and Tigger DNA transposons. Moreover, SAFB also suppresses LTR/ERV elements in species in which they are still active, such as mice and flies. A significant subset of splicing events suppressed by SAFB in somatic cells are activated in the testis, coinciding with low SAFB expression in postmeiotic spermatids. Reminiscent of the division of labour between innate and adaptive immune systems that fight external pathogens, our results uncover SAFB proteins as an RNA-based, pattern-guided, non-adaptive defence system against TEs in the soma, complementing the RNA-based, adaptive Piwi-interacting RNA pathway of the germline.


Assuntos
Elementos de DNA Transponíveis , Íntrons , Precursores de RNA , Splicing de RNA , RNA Mensageiro , Animais , Humanos , Masculino , Camundongos , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Éxons/genética , Genoma/genética , Íntrons/genética , Especificidade de Órgãos/genética , RNA de Interação com Piwi/genética , RNA de Interação com Piwi/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermátides/citologia , Espermátides/metabolismo , Splicing de RNA/genética , Testículo , Meiose
11.
BMC Plant Biol ; 24(1): 88, 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38317087

RESUMO

Mounting evidence recognizes structural variations (SVs) and repetitive DNA sequences as crucial players in shaping the existing grape phenotypic diversity at intra- and inter-species levels. To deepen our understanding on the abundance, diversity, and distribution of SVs and repetitive DNAs, including transposable elements (TEs) and tandemly repeated satellite DNA (satDNAs), we re-sequenced the genomes of the ancient grapes Aglianico and Falanghina. The analysis of large copy number variants (CNVs) detected candidate polymorphic genes that are involved in the enological features of these varieties. In a comparative analysis of Aglianico and Falanghina sequences with 21 publicly available genomes of cultivated grapes, we provided a genome-wide annotation of grape TEs at the lineage level. We disclosed that at least two main clusters of grape cultivars could be identified based on the TEs content. Multiple TEs families appeared either significantly enriched or depleted. In addition, in silico and cytological analyses provided evidence for a diverse chromosomal distribution of several satellite repeats between Aglianico, Falanghina, and other grapes. Overall, our data further improved our understanding of the intricate grape diversity held by two Italian traditional varieties, unveiling a pool of unique candidate genes never so far exploited in breeding for improved fruit quality.


Assuntos
Vitis , Humanos , Vitis/genética , Melhoramento Vegetal , Elementos de DNA Transponíveis/genética , DNA Satélite
12.
PLoS One ; 19(2): e0294570, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38349924

RESUMO

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a global burden for livestock producers and has an association with Crohn's disease in humans. Within MAP there are two major lineages, S/Type I/TypeIII and C/Type II, that vary in phenotype including culturability, host preference and virulence. These lineages have been identified using the IS1311 element, which contains a conserved, single nucleotide polymorphism. IS1311 and the closely related IS1245 element belong to the IS256 family of insertion sequences, are dispersed throughout M. avium taxa but remain poorly characterised. To investigate the distribution and diversity of IS1311 in MAP, 805 MAP genomes were collated from public databases. IS1245 was absent, while IS1311 sequence, copy number and insertion loci were conserved between MAP S lineages and varied within the MAP C lineage. One locus was specific to the S strains, which contained nine IS1311 copies. In contrast, C strains contained either seven or eight IS1311 loci. Most insertion loci were associated with the boundaries of homologous regions that had undergone genome rearrangement between the MAP lineages, suggesting that this sequence may be a driver of recombination. Phylogenomic geographic clustering of MAP subtypes was demonstrated for the first time, at continental scale, and indicated that there may have been recent MAP transmission between Europe and North America, in contrast to Australia where importation of live ruminants is generally prohibited. This investigation confirmed the utility of IS1311 typing in epidemiological studies and resolved anomalies in past studies. The results shed light on potential mechanisms of niche/host adaptation, virulence of MAP and global transmission dynamics.


Assuntos
Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Humanos , Mycobacterium avium subsp. paratuberculosis/genética , Adaptação ao Hospedeiro , Paratuberculose/microbiologia , Polimorfismo de Nucleotídeo Único , Ruminantes/genética , Elementos de DNA Transponíveis
13.
Nat Commun ; 15(1): 1701, 2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38402218

RESUMO

The spatial organization of eukaryotic genomes is linked to their biological functions, although it is not clear how this impacts the overall evolution of a genome. Here, we uncover the three-dimensional (3D) genome organization of the phytopathogen Verticillium dahliae, known to possess distinct genomic regions, designated adaptive genomic regions (AGRs), enriched in transposable elements and genes that mediate host infection. Short-range DNA interactions form clear topologically associating domains (TADs) with gene-rich boundaries that show reduced levels of gene expression and reduced genomic variation. Intriguingly, TADs are less clearly insulated in AGRs than in the core genome. At a global scale, the genome contains bipartite long-range interactions, particularly enriched for AGRs and more generally containing segmental duplications. Notably, the patterns observed for V. dahliae are also present in other Verticillium species. Thus, our analysis links 3D genome organization to evolutionary features conserved throughout the Verticillium genus.


Assuntos
Genômica , Plantas , Plantas/genética , Elementos de DNA Transponíveis/genética , Cromatina/genética , Evolução Molecular , Genoma Fúngico/genética
14.
Genome Biol Evol ; 16(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38368625

RESUMO

The clouded apollo (Parnassius mnemosyne) is a palearctic butterfly distributed over a large part of western Eurasia, but population declines and fragmentation have been observed in many parts of the range. The development of genomic tools can help to shed light on the genetic consequences of the decline and to make informed decisions about direct conservation actions. Here, we present a high-contiguity, chromosome-level genome assembly of a female clouded apollo butterfly and provide detailed annotations of genes and transposable elements. We find that the large genome (1.5 Gb) of the clouded apollo is extraordinarily repeat rich (73%). Despite that, the combination of sequencing techniques allowed us to assemble all chromosomes (nc = 29) to a high degree of completeness. The annotation resulted in a relatively high number of protein-coding genes (22,854) compared with other Lepidoptera, of which a large proportion (21,635) could be assigned functions based on homology with other species. A comparative analysis indicates that overall genome structure has been largely conserved, both within the genus and compared with the ancestral lepidopteran karyotype. The high-quality genome assembly and detailed annotation presented here will constitute an important tool for forthcoming efforts aimed at understanding the genetic consequences of fragmentation and decline, as well as for assessments of genetic diversity, population structure, inbreeding, and genetic load in the clouded apollo butterfly.


Assuntos
Borboletas , Animais , Feminino , Borboletas/genética , Conservação dos Recursos Naturais , Genômica , Elementos de DNA Transponíveis , Cromossomos , Anotação de Sequência Molecular
15.
Cells ; 13(3)2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38334619

RESUMO

Endogenous double-stranded RNA has emerged as a potent stimulator of innate immunity. Under physiological conditions, endogenous dsRNA is maintained in the cell nucleus or the mitochondria; however, if protective mechanisms are breached, it leaches into the cytoplasm and triggers immune signaling pathways. Ectopic activation of innate immune pathways is associated with various diseases and senescence and can trigger apoptosis. Hereby, the level of cytoplasmic dsRNA is crucial. We have enriched dsRNA from two melanoma cell lines and primary dermal fibroblasts, including a competing probe, and analyzed the dsRNA transcriptome using RNA sequencing. There was a striking difference in read counts between the cell lines and the primary cells, and the effect was confirmed by northern blotting and immunocytochemistry. Both mitochondria (10-20%) and nuclear transcription (80-90%) contributed significantly to the dsRNA transcriptome. The mitochondrial contribution was lower in the cancer cells compared to fibroblasts. The expression of different transposable element families was comparable, suggesting a general up-regulation of transposable element expression rather than stimulation of a specific sub-family. Sequencing of the input control revealed minor differences in dsRNA processing pathways with an upregulation of oligoadenylate synthase and RNP125 that negatively regulates the dsRNA sensors RIG1 and MDA5. Moreover, RT-qPCR, Western blotting, and immunocytochemistry confirmed the relatively minor adaptations to the hugely different dsRNA levels. As a consequence, these transformed cell lines are potentially less tolerant to interventions that increase the formation of endogenous dsRNA.


Assuntos
Elementos de DNA Transponíveis , RNA de Cadeia Dupla , Células Cultivadas , Imunidade Inata/genética , Linhagem Celular
16.
Genome Biol ; 25(1): 52, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38378611

RESUMO

BACKGROUND: Centromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of assembling repetitive genomic regions. RESULTS: Using recently available PacBio long-read sequencing data from the C57BL/6 strain, we find that contrary to the previous reports of their homogeneous nature, both centromeric minor satellites and pericentromeric major satellites exhibit a high degree of variation in sequence and organization within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Using chromatin immunoprecipitation sequencing (ChIP-seq), we find that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence enrichment and homogeneity at these regions. The transposable elements at centromeric regions are not part of functional centromeres as they lack significant CENP-A enrichment. Furthermore, both CENP-A and H3K9me3 nucleosomes occupy minor and major satellites spanning centromeric-pericentric junctions and a low yet significant amount of CENP-A spreads locally at centromere junctions on both pericentric and telocentric sides. Finally, while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays are poorly phased. Interestingly, the homogeneous class of major satellites also phase CENP-A and H3K27me3 nucleosomes, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites. CONCLUSIONS: Our findings reveal that mouse centromeres and pericentromeres display a high diversity in satellite sequence, organization, and chromatin structure.


Assuntos
Elementos de DNA Transponíveis , Nucleossomos , Camundongos , Animais , Proteína Centromérica A/genética , Camundongos Endogâmicos C57BL , Centrômero , Cromatina , DNA Satélite , Autoantígenos
17.
Cancer Res ; 84(6): 808-826, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38345497

RESUMO

Heterochromatin loss and genetic instability enhance cancer progression by favoring clonal diversity, yet uncontrolled replicative stress leads to mitotic catastrophe and inflammatory responses that promote immune rejection. KRAB domain-containing zinc finger proteins (KZFP) contribute to heterochromatin maintenance at transposable elements (TE). Here, we identified an association of upregulation of a cluster of primate-specific KZFPs with poor prognosis, increased copy-number alterations, and changes in the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL). Depleting two of these KZFPs targeting evolutionarily recent TEs, ZNF587 and ZNF417, impaired the proliferation of cells derived from DLBCL and several other tumor types. ZNF587 and ZNF417 depletion led to heterochromatin redistribution, replicative stress, and cGAS-STING-mediated induction of an interferon/inflammatory response, which enhanced susceptibility to macrophage-mediated phagocytosis and increased surface expression of HLA-I, together with presentation of a neoimmunopeptidome. Thus, cancer cells can exploit KZFPs to dampen TE-originating surveillance mechanisms, which likely facilitates clonal expansion, diversification, and immune evasion. SIGNIFICANCE: Upregulation of a cluster of primate-specific KRAB zinc finger proteins in cancer cells prevents replicative stress and inflammation by regulating heterochromatin maintenance, which could facilitate the development of improved biomarkers and treatments.


Assuntos
Heterocromatina , Neoplasias , Animais , Heterocromatina/genética , Dedos de Zinco/genética , Elementos de DNA Transponíveis , Primatas/genética , Inflamação/genética , Neoplasias/genética
18.
Nucleic Acids Res ; 52(5): 2724-2739, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38300794

RESUMO

Transposons, as non-viral integration vectors, provide a secure and efficient method for stable gene delivery. In this study, we have discovered Mage (MG), a novel member of the piggyBac(PB) family, which exhibits strong transposability in a variety of mammalian cells and primary T cells. The wild-type MG showed a weaker insertion preference for near genes, transcription start sites (TSS), CpG islands, and DNaseI hypersensitive sites in comparison to PB, approaching the random insertion pattern. Utilizing in silico virtual screening and feasible combinatorial mutagenesis in vitro, we effectively produced the hyperactive MG transposase (hyMagease). This variant boasts a transposition rate 60% greater than its native counterpart without significantly altering its insertion pattern. Furthermore, we applied the hyMagease to efficiently deliver chimeric antigen receptor (CAR) into T cells, leading to stable high-level expression and inducing significant anti-tumor effects both in vitro and in xenograft mice models. These findings provide a compelling tool for gene transfer research, emphasizing its potential and prospects in the domains of genetic engineering and gene therapy.


Assuntos
Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Humanos , Camundongos , Animais , Elementos de DNA Transponíveis/genética , Terapia Genética , Linfócitos T/metabolismo , Transposases/genética , Transposases/metabolismo , Vetores Genéticos , Mamíferos/genética
19.
PLoS Biol ; 22(2): e3002505, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38363809

RESUMO

Alternative splicing is an essential regulatory mechanism for development and pathogenesis. Through alternative splicing one gene can encode multiple isoforms and be translated into proteins with different functions. Therefore, this diversity is an important dimension to understand the molecular mechanism governing embryo development. Isoform expression in preimplantation embryos has been extensively investigated, leading to the discovery of new isoforms. However, the dynamics of isoform switching of different types of transcripts throughout the development remains unexplored. Here, using single-cell direct isoform sequencing in over 100 single blastomeres from the mouse oocyte to blastocyst stage, we quantified isoform expression and found that 3-prime partial transcripts lacking stop codons are highly accumulated in oocytes and zygotes. These transcripts are not transcription by-products and might play a role in maternal to zygote transition (MZT) process. Long-read sequencing also enabled us to determine the expression of transposable elements (TEs) at specific loci. In this way, we identified 3,894 TE loci that exhibited dynamic changes along the preimplantation development, likely regulating the expression of adjacent genes. Our work provides novel insights into the transcriptional regulation of early embryo development.


Assuntos
Elementos de DNA Transponíveis , Desenvolvimento Embrionário , Feminino , Gravidez , Animais , Camundongos , Elementos de DNA Transponíveis/genética , Desenvolvimento Embrionário/genética , Isoformas de Proteínas/genética , Zigoto , Análise de Célula Única
20.
Sci Rep ; 14(1): 4723, 2024 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-38413664

RESUMO

Z-DNA, a well-known non-canonical form of DNA involved in gene regulation, is often found in gene promoters. Transposable elements (TEs), which make up 45% of the human genome, can move from one location to another within the genome. TEs play various biological roles in host organisms, and like Z-DNA, can influence transcriptional regulation near promoter regions. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a critical role in the regulation of gene expression. Although TEs can generate Z-DNA and miRNAs can bind to Z-DNA, how these factors affect gene transcription has yet to be elucidated. Here, we identified potential Z-DNA forming sequence (ZFS), including TE-derived ZFS, in the promoter of prostaglandin reductase 1 (PTGR1) by data analysis. The transcriptional activity of these ZFS in PTGR1 was confirmed using dual-luciferase reporter assays. In addition, we discovered a novel ZFS-binding miRNA (miR-6867-5p) that suppressed PTGR1 expression by targeting to ZFS. In conclusion, these findings suggest that ZFS, including TE-derived ZFS, can regulate PTGR1 gene expression and that miR-6867-5p can suppress PTGR1 by interacting with ZFS.


Assuntos
DNA Forma Z , MicroRNAs , Humanos , Elementos de DNA Transponíveis/genética , Expressão Gênica , Regulação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo
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