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1.
Mol Genet Genomics ; 295(1): 47-54, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31420737

RESUMO

Stem trichomes and seed fibers originate from epidermal cells and partially share a regulatory pathway at the molecular level. In Gossypium barbadense, two insertions of a Ty1 long-terminal repeat-retrotransposon [transposable element TE1 and TE2] in a homeodomain-leucine zipper gene (HD1) result in glabrous stems. The primers used to identify the TE insertions in G. barbadense were applied to screen for the same events in 81 modern G. hirsutum varieties and 31 wild races. Three wild races were found carrying the same TEs as G. barbadense. However, the TE insertions in two of these wild races occurred at different sites (4th exon), therefore, named TE3, while the TE in the other wild race occurred at the same site as TE2. An RNA sequencing and qRT-PCR analysis indicated that the loss of HD1 function was caused by the TE insertion. Genetic mapping revealed a strong association between glabrous stems and TE3 insertions, confirming that HD1 is a critical gene for stem trichome initiation in G. hirsutum, as in G. barbadense. Using the long-terminal repeat sequence as a query to search against the Texas Marker-1 reference genome sequence, we found that the TE occurred after tetraploid cotton formation and evolved at different rates in G. hirsutum and G. barbadense. Interestingly, at least three independent insertion events of the same retrotransposon occurred preferentially in the A sub-genome's HD1 gene, but not in the D sub-genome of G. hirsutum or G. barbadense, suggesting that an unknown TE insertion mechanism and resultant gene function changes may have hastened cotton speciation.


Assuntos
Proteínas de Arabidopsis/genética , Gossypium/genética , Histona Desacetilases/genética , Mutagênese Insercional/genética , Caules de Planta/genética , Retroelementos/genética , Sequências Repetidas Terminais/genética , Tricomas/genética , Mapeamento Cromossômico/métodos , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta/genética , Zíper de Leucina/genética , Fenótipo , Filogenia , Tetraploidia
2.
BMC Bioinformatics ; 20(Suppl 9): 484, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757208

RESUMO

BACKGROUND: Transposable Elements (TE) are mobile sequences that make up large portions of eukaryote genomes. The functions they play within the complex cellular architecture are still not clearly understood, but it is becoming evident that TE have a role in several physiological and pathological processes. In particular, it has been shown that TE transcription is necessary for the correct development of mice embryos and that their expression is able to finely modulate transcription of coding and non-coding genes. Moreover, their activity in the central nervous system (CNS) and other tissues has been correlated with the creation of somatic mosaicisms and with pathologies such as neurodevelopmental and neurodegenerative diseases as well as cancers. RESULTS: We analyzed TE expression among different cell types of the Caenorhabditis elegans (C. elegans) early embryo asking if, where and when TE are expressed and whether their expression is correlated with genes playing a role in early embryo development. To answer these questions, we took advantage of a public C. elegans embryonic single-cell RNA-seq (sc-RNAseq) dataset and developed a bioinformatics pipeline able to quantify reads mapping specifically against TE, avoiding counting reads mapping on TE fragments embedded in coding/non-coding transcripts. Our results suggest that i) canonical TE expression analysis tools, which do not discard reads mapping on TE fragments embedded in annotated transcripts, may over-estimate TE expression levels, ii) Long Terminal Repeats (LTR) elements are mostly expressed in undifferentiated cells and might play a role in pluripotency maintenance and activation of the innate immune response, iii) non-LTR are expressed in differentiated cells, in particular in neurons and nervous system-associated tissues, and iv) DNA TE are homogenously expressed throughout the C. elegans early embryo development. CONCLUSIONS: TE expression appears finely modulated in the C. elegans early embryo and different TE classes are expressed in different cell types and stages, suggesting that TE might play diverse functions during early embryo development.


Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Elementos de DNA Transponíveis/genética , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Linhagem da Célula/genética , Biologia Computacional , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/genética , Imunidade Inata/genética , Células-Tronco Pluripotentes/metabolismo , Sequências Repetidas Terminais/genética
3.
Postepy Biochem ; 65(3): 217-223, 2019 10 01.
Artigo em Polonês | MEDLINE | ID: mdl-31643169

RESUMO

Transposable elements (TEs) are the sequences that are able to "jump" across the genome. They are found in virtually all organisms including human. Although in human, the majority of TEs lost their ability to autonomous transposition, they make up almost half of our genome, and played important roles in genome evolution. Fast progress in deep sequencing and functional analysis has revealed the importance of domes­ticated copies of transposable elements, including their regulatory sequences, transcripts and proteins in normal cells functioning. However, a growing numer of evidence suggest the involvment of TEs in development and progression of autoimmune and neurodegenerative disaeses as well as in many types of cancer. In this review we summarize the current state of knowledge about the LTR retroelements: endogenous retroviruses (ERVs) and Ty3/Gypsy retrotransposons, and their role in human organism.


Assuntos
Genoma Humano/genética , Retroelementos/genética , Doenças Autoimunes/genética , Retrovirus Endógenos/genética , Evolução Molecular , Humanos , Neoplasias/genética , Doenças Neurodegenerativas/genética , Sequências Repetidas Terminais/genética
4.
Planta ; 250(5): 1781-1787, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31562541

RESUMO

MAIN CONCLUSION: Copia/Ale is the youngest lineage in both Solanum tuberosum and S. commersonii. Within it, we identified nightshade, a new LTR element active in the cultivated potato. From an evolutionary perspective, long-terminal repeat retrotransposons (LTR-RT) activity during stress may be viewed as a mean by which organisms can keep up rates of genetic adaptation to changing conditions. Potato is one of the most important crop consumed worldwide, but studies on LTR-RT characterization are still lacking. Here, we assessed the abundance, insertion time and activity of LTR-RTs in both cultivated Solanum tuberosum and its cold-tolerant wild relative S. commersonii genomes. Gypsy elements were more abundant than Copia ones, suggesting that the former was somehow more successful in colonizing potato genomes. However, Copia elements, and in particular, the Ale lineage, are younger than Gypsy ones, since their insertion time was in average ~ 2 Mya. Due to the ability of LTR-RTs to be circularized by the host DNA repair mechanisms, we identified via mobilome-seq a Copia/Ale element (called nightshade, informal name used for potato family) active in S. tuberosum genome. Our analyses represent a valuable resource for comparative genomics within the Solanaceae, transposon-tagging and for the design of cultivar-specific molecular markers in potato.


Assuntos
Genoma de Planta/genética , Genômica , Retroelementos/ética , Solanum/genética , Sequências Repetidas Terminais/genética , Temperatura Baixa , Evolução Molecular , Marcadores Genéticos/genética , Solanum/fisiologia , Solanum tuberosum/genética , Solanum tuberosum/fisiologia , Estresse Fisiológico
5.
PLoS Genet ; 15(9): e1008370, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525177

RESUMO

Transposable elements in crop plants are the powerful drivers of phenotypic variation that has been selected during domestication and breeding programs. In tomato, transpositions of the LTR (long terminal repeat) retrotransposon family Rider have contributed to various phenotypes of agronomical interest, such as fruit shape and colour. However, the mechanisms regulating Rider activity are largely unknown. We have developed a bioinformatics pipeline for the functional annotation of retrotransposons containing LTRs and defined all full-length Rider elements in the tomato genome. Subsequently, we showed that accumulation of Rider transcripts and transposition intermediates in the form of extrachromosomal DNA is triggered by drought stress and relies on abscisic acid signalling. We provide evidence that residual activity of Rider is controlled by epigenetic mechanisms involving siRNAs and the RNA-dependent DNA methylation pathway. Finally, we demonstrate the broad distribution of Rider-like elements in other plant species, including crops. Our work identifies Rider as an environment-responsive element and a potential source of genetic and epigenetic variation in plants.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Lycopersicon esculentum/genética , Retroelementos/genética , Biologia Computacional/métodos , Epigênese Genética/genética , Evolução Molecular , Genes de Plantas/genética , Genoma de Planta/genética , Lycopersicon esculentum/crescimento & desenvolvimento , Análise de Sequência de DNA/métodos , Sequências Repetidas Terminais/genética
6.
PLoS One ; 14(5): e0214542, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31107873

RESUMO

Long terminal repeat retrotransposons (LTR-RTs) in plant genomes differ in abundance, structure and genomic distribution, reflecting the large number of evolutionary lineages. Elements within lineages can be considered populations, in which each element is an individual in its genomic environment. In this way, it would be reasonable to apply microevolutionary analyses to understand transposable element (TE) evolution, such as those used to study the genetic structure of natural populations. Here, we applied a Bayesian method to infer genetic structure of populations together with classical phylogenetic and dating tools to analyze LTR-RT evolution using the monocot Setaria italica as a model species. In contrast to a phylogeny, the Bayesian clusterization method identifies populations by assigning individuals to one or more clusters according to the most probabilistic scenario of admixture, based on genetic diversity patterns. In this work, each LTR-RT insertion was considered to be one individual and each LTR-RT lineage was considered to be a single species. Nine evolutionary lineages of LTR-RTs were identified in the S. italica genome that had different genetic structures with variable numbers of clusters and levels of admixture. Comprehensive analysis of the phylogenetic, clusterization and time of insertion data allowed us to hypothesize that admixed elements represent sequences that harbor ancestral polymorphic sequence signatures. In conclusion, application of microevolutionary concepts in genome evolution studies is suitable as a complementary approach to phylogenetic analyses to address the evolutionary history and functional features of TEs.


Assuntos
Evolução Molecular , Genética Populacional , Retroelementos/genética , Setaria (Planta)/genética , Sequências Repetidas Terminais/genética , Teorema de Bayes , Ligação Genética , Genoma de Planta , Filogenia , Setaria (Planta)/classificação
7.
PLoS Biol ; 17(5): e3000241, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31086362

RESUMO

Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.


Assuntos
Centrômero/genética , Drosophila/genética , Retroelementos/genética , Animais , Proteína Centromérica A/genética , Cromatina/metabolismo , Elementos de DNA Transponíveis/genética , DNA Satélite/genética , Drosophila/embriologia , Proteínas de Drosophila/genética , Embrião não Mamífero/metabolismo , Genoma de Inseto , Sequências Repetidas Terminais/genética
8.
Genes (Basel) ; 10(5)2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064091

RESUMO

Transposable elements (TEs) are ubiquitous in arthropods. However, analyses of large-scale and long-term coevolution between TEs and host genomes remain scarce in arthropods. Here, we choose 14 representative Arthropoda species from eight orders spanning more than 500 million years of evolution. By developing an unbiased TE annotation pipeline, we obtained 87 to 2266 TE reference sequences in a species, which is a considerable improvement compared to the reference TEs previously annotated in Repbase. We find that TE loads are diversified among species and were previously underestimated. The highly species- and time-specific expansions and contractions, and intraspecific sequence diversification are the leading driver of long terminal repeat (LTR) dynamics in Lepidoptera. Terminal inverted repeats (TIRs) proliferated substantially in five species with large genomes. A phylogenetic comparison reveals that the loads of multiple TE subfamilies are positively correlated with genome sizes. We also identified a few horizontally transferred TE candidates across nine species. In addition, we set up the Arthropod Transposable Elements database (ArTEdb) to provide TE references and annotations. Collectively, our results provide high-quality TE references and uncover that TE loads and expansion histories vary greatly among arthropods, which implies that TEs are an important driving force shaping the evolution of genomes through gain and loss.


Assuntos
Artrópodes/genética , Elementos de DNA Transponíveis/genética , Evolução Molecular , Genoma/genética , Animais , Tamanho do Genoma , Anotação de Sequência Molecular , Filogenia , Sequências Repetidas Terminais/genética
9.
Genes (Basel) ; 10(4)2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970574

RESUMO

The evolutionary dynamics of long terminal repeat (LTR) retrotransposons in tree genomes has remained largely unknown. The availability of the complete genome sequences of the mulberry tree (Morus notabilis) has offered an unprecedented opportunity for us to characterize these retrotransposon elements. We investigated 202 and 114 families of Copia and Gypsy superfamilies, respectively, comprising 2916 intact elements in the mulberry genome. The tRNAMet was the most frequently used type of tRNA in both superfamilies. Phylogenetic analysis suggested that Copia and Gypsy from mulberry can be grouped into eight and six lineages, respectively. All previously characterized families of such elements could also be found in the mulberry genome. About 95% of the identified Copia and Gypsy full elements were estimated to have been inserted into the mulberry genome within the past 2­3 million years. Meanwhile, the estimated insertion times of members of the three most abundant families of the Copia superfamily (908 members from the three most abundant families) and Gypsy superfamily (783 members from the three most abundant families) revealed divergent life histories. Compared with the situation in Gypsy elements, three families of Copia elements are under positive selection pressure, which suggested that Copia elements may have a dominant influence in the evolution of mulberry genes. Analysis of insertion and deletion dynamics suggested that Copia and Gypsy elements exhibited a very long half-life in the mulberry genome. The present work provides new insights into the insertion and deletion dynamics of LTR retrotransposons, and it will greatly improve our understanding of the important roles transposable elements play in the architecture of the mulberry genome.


Assuntos
Evolução Molecular , Morus/genética , Retroelementos/genética , Sequências Repetidas Terminais/genética , Genoma de Planta/genética , Filogenia , Especificidade da Espécie
10.
BMC Plant Biol ; 19(1): 140, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987586

RESUMO

BACKGROUND: Miniature inverted-repeat transposable elements (MITEs) and long terminal repeat (LTR) retrotransposons are ubiquitous in plants genomes, and highly important in their evolution and diversity. However, their mechanisms of insertion/amplification and roles in Citrus genome's evolution/diversity are still poorly understood. RESULTS: To address this knowledge gap, we developed different computational pipelines to analyze, annotate and classify MITEs and LTR retrotransposons in six different sequenced Citrus species. We identified 62,010 full-length MITEs from 110 distinguished families. We observed MITEs tend to insert in gene related regions and enriched in promoters. We found that DTM63 is possibly an active Mutator-like MITE family in the traceable past and may still be active in Citrus. The insertion of MITEs resulted in massive polymorphisms and played an important role in Citrus genome diversity and gene structure variations. In addition, 6630 complete LTR retrotransposons and 13,371 solo-LTRs were identified. Among them, 12 LTR lineages separated before the differentiation of mono- and dicotyledonous plants. We observed insertion and deletion of LTR retrotransposons was accomplished with a dynamic balance, and their half-life in Citrus was ~ 1.8 million years. CONCLUSIONS: These findings provide insights into MITEs and LTR retrotransposons and their roles in genome diversity in different Citrus genomes.


Assuntos
Citrus/genética , Elementos de DNA Transponíveis/genética , Genoma de Planta/genética , Sequências Repetidas Invertidas/genética , Retroelementos/genética , Sequências Repetidas Terminais/genética , Variação Genética
11.
PLoS One ; 14(4): e0213770, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30934003

RESUMO

Human endogenous retroviruses (HERVs) have been investigated for potential links with human cancer. However, the distribution of somatic nucleotide variations in HERV elements has not been explored in detail. This study aims to identify HERV elements with an over-representation of somatic mutations (hot spots) in cancer patients. Four HERV elements with mutation hotspots were identified that overlap with exons of four human protein coding genes. These hotspots were identified based on the significant over-representation (p<8.62e-4) of non-synonymous single-nucleotide variations (nsSNVs). These genes are TNN (HERV-9/LTR12), OR4K15 (HERV-IP10F/LTR10F), ZNF99 (HERV-W/HERV17/LTR17), and KIR2DL1 (MST/MaLR). In an effort to identify mutations that effect survival, all nsSNVs were further evaluated and it was found that kidney cancer patients with mutation C2270G in ZNF99 have a significantly lower survival rate (hazard ratio = 2.6) compared to those without it. Among HERV elements in the human non-protein coding regions, we found 788 HERVs with significantly elevated numbers of somatic single-nucleotide variations (SNVs) (p<1.60e-5). From this category the top three HERV elements with significantly over-represented SNVs are HERV-H/LTR7, HERV-9/LTR12 and HERV-L/MLT2. Majority of the SNVs in these 788 HERV elements are located in three DNA functional groups: long non-coding RNAs (lncRNAs) (60%), introns (22.2%) and transcriptional factor binding sites (TFBS) (14.8%). This study provides a list of mutational hotspots in HERVs, which could potentially be used as biomarkers and therapeutic targets.


Assuntos
Retrovirus Endógenos/genética , Genoma Humano/genética , Neoplasias Renais/genética , Polimorfismo de Nucleotídeo Único/genética , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Íntrons/genética , Neoplasias Renais/patologia , Mutação , RNA Longo não Codificante/genética , Receptores KIR2DL1/genética , Análise de Sobrevida , Tenascina/genética , Sequências Repetidas Terminais/genética
12.
EBioMedicine ; 41: 427-442, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827930

RESUMO

BACKGROUND: Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior. METHODS: We performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA). FINDINGS: Though HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC. INTERPRETATION: Integrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs.


Assuntos
Retrovirus Endógenos/genética , Epigenômica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Redutases do Citocromo/genética , Retrovirus Endógenos/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Proteínas/genética , Pirofosfatases/genética , Taxa de Sobrevida , Sequências Repetidas Terminais/genética , Enzimas de Conjugação de Ubiquitina/genética
13.
Arch Virol ; 164(5): 1459-1467, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30880347

RESUMO

In this study, we describe a novel and rapid method for the construction of a full-length infectious clone (pPPV). The constructed clone contained an engineered EcoRv site that served as a genetic marker and was shown to be infectious when transfected into a monolayer of PK-15 cells. The rescued virus (rPPV) of the infectious clone was found to be indistinguishable from wild-type virus BQ in terms of its biological properties. The generation of this PPV infectious clone provides a potentially powerful tool with which to elucidate the molecular pathogenesis of PPV.


Assuntos
Clonagem Molecular/métodos , Genoma Viral/genética , Parvovirus Suíno/genética , Sequências Repetidas Terminais/genética , Animais , Linhagem Celular , Técnica Indireta de Fluorescência para Anticorpo , Marcadores Genéticos/genética , Técnicas de Amplificação de Ácido Nucleico , Suínos
14.
BMC Genomics ; 20(1): 25, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30626325

RESUMO

BACKGROUND: Orchids produce a colorless protocorm by symbiosis with fungi upon seed germination. For mass production of orchids, the prevailing approaches are both generation of protocorm-like bodies (PLBs) from callus and multiplication of adventitious buds on inflorescence. However, somaclonal variations occur during micropropagation. RESULTS: We isolated the two most expressed transposable elements belonging to P Instability Factor (PIF)-like transposons. Among them, a potential autonomous element was identified by similarity analysis against the whole-genome sequence of Phalaenopsis equestris and named PePIF1. It contains a 19-bp terminal inverted repeat flanked by a 3-bp target site duplication and two coding regions encoding ORF1- and transposase-like proteins. Phylogenetic analysis revealed that PePIF1 belongs to a new P-lineage of PIF. Furthermore, two distinct families, PePIF1a and PePIF1b, with 29 and 37 putative autonomous elements, respectively, were isolated, along with more than 3000 non-autonomous and miniature inverted-repeat transposable element (MITE)-like elements. Among them, 828 PePIF1-related elements were inserted in 771 predicted genes. Intriguingly, PePIF1 was transposed in the somaclonal variants of Phalaenopsis cultivars, as revealed by transposon display, and the newly inserted genes were identified and sequenced. CONCLUSION: A PIF-like element, PePIF1, was identified in the Phalaenopsis genome and actively transposed during micropropagation. With the identification of PePIF1, we have more understanding of the Phalaenopsis genome structure and somaclonal variations during micropropagation for use in orchid breeding and production.


Assuntos
Elementos de DNA Transponíveis/genética , Orchidaceae/genética , Filogenia , Genoma de Planta/genética , Mutagênese Insercional/genética , Fases de Leitura Aberta , Sequências Repetidas Terminais/genética , Transposases/genética
15.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626680

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates episome persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence through its carboxy-terminal domain to mediate DNA replication. LANA simultaneously binds mitotic chromosomes and TR DNA to segregate virus genomes to daughter cell nuclei. Amino-terminal LANA attaches to chromosomes by binding histones H2A/H2B, and carboxy-terminal LANA contributes to mitotic-chromosome binding. Although amino- and carboxy-terminal LANA are essential for episome persistence, they are not sufficient, since deletion of all internal LANA sequence renders LANA highly deficient for episome maintenance. Internal LANA sequence upstream of the internal repeat elements contributes to episome segregation and persistence. Here, we investigate this region with a panel of LANA deletion mutants. Mutants retained the ability to associate with mitotic chromosomes and bind TR DNA. In contrast to prior results, deletion of most of this sequence did not reduce LANA's ability to mediate DNA replication. Deletions of upstream sequence within the region compromised segregation of TR DNA to daughter cells, as assessed by retention of green fluorescent protein (GFP) expression from a replication-deficient TR plasmid. However, deletion of this upstream sequence did not reduce episome maintenance. In contrast, deletions that included an 80-amino-acid sequence immediately downstream resulted in highly deficient episome persistence. LANA with this downstream sequence deleted maintained the ability to replicate and segregate TR DNA, suggesting a unique role for the residues. Therefore, this work identifies adjacent LANA regions with distinct roles in episome segregation and persistence.IMPORTANCE KSHV LANA mediates episomal persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence to mediate DNA replication and tethers KSHV DNA to mitotic chromosomes to segregate genomes to daughter cell nuclei. Here, we investigate LANA sequence upstream of the internal repeat elements that contributes to episome segregation and persistence. Mutants with deletions within this sequence maintained the ability to bind mitotic chromosomes or bind and replicate TR DNA. Deletion of upstream sequence within the region reduced segregation of TR DNA to daughter cells, but not episome maintenance. In contrast, mutants with deletions of 80 amino acids immediately downstream were highly deficient for episome persistence yet maintained the ability to replicate and segregate TR DNA, the two principal components of episome persistence, suggesting another role for the residues. In summary, this work identifies adjacent LANA sequence with distinct roles in episome segregation and persistence.


Assuntos
Antígenos Virais/genética , Herpesvirus Humano 8/genética , Proteínas Nucleares/genética , Plasmídeos/genética , Sarcoma de Kaposi/virologia , Antígenos Nucleares/genética , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/virologia , Cromossomos/genética , Replicação do DNA/genética , DNA Viral/genética , Genoma Viral/genética , Células HEK293 , Humanos , Mitose/genética , Sequências Repetidas Terminais/genética , Proteínas Virais/genética , Latência Viral/genética , Replicação Viral/genética
16.
J Virol ; 93(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651368

RESUMO

Latency-associated nuclear antigen (LANA) is essential for maintaining the viral genome by regulating replication and segregation of the viral episomes. The virus maintains 50 to 100 episomal copies during latency and replicates in synchrony with the cellular DNA of the infected cells. Since virus lacks its own replication machinery, it utilizes the cellular proteins for replication and maintenance, and LANA has been shown to make many of these proteins available for replication by directly recruiting them to the viral origin of replication within the terminal repeat (TR) region. Our studies identified members of the minichromosome maintenance (MCM) complex as potential LANA-interacting proteins. Here, we show that LANA specifically interacts with the components of the MCM complex, primarily during the G1/S phase of the cell cycle. MCM3 and -4 of the MCM complex specifically bound to the amino-terminal domain, while MCM6 bound to both the amino- and carboxyl-terminal domains of LANA. The MCM binding region in the N-terminal domain mapped to the chromatin binding domain (CBD). LANA with point mutations in the carboxyl-terminal domain identified an MCM6 binding domain, and overexpression of that domain (amino acids [aa] 1100 to 1150) abolished TR replication. Introduction of a peptide encompassing the LANA aa 1104 to 1123 reduced MCM6 association with LANA and TR replication. Moreover, a recombinant Kaposi's sarcoma-associated herpesvirus (KSHV) expressing LANA with a deletion of aa 1100 to 1150 (BAC16Δ1100-1150, where BAC is bacmid) showed reduced replication and persistence of viral genome copies compared to levels with the wild-type BAC16. Additionally, the role of MCMs in viral replication was confirmed by depleting MCMs and assaying transient and long-term maintenance of the viral episomes. The recruitment of MCMs to the replication origins through LANA was demonstrated through chromatin immunoprecipitation and isolation of proteins on nascent replicated DNA (iPOND). These data clearly show the role of MCMs in latent DNA replication and the potential for targeting the C-terminal domain of LANA to block viral persistence.IMPORTANCE LANA-mediated latent DNA replication is essential for efficient maintenance of KSHV episomes in the host. During latency, virus relies on the host cellular machinery for replication, which occurs in synchrony with the cellular DNA. LANA interacts with the components of multiple cellular pathways, including cellular replication machinery, and recruits them to the viral origin for DNA replication. In this study, we characterize the interactions between LANA and minichromosome maintenance (MCM) proteins, members of the cellular replication complex. We demonstrated a cell cycle-dependent interaction between LANA and MCMs and determined their importance for viral genome replication and maintenance through biochemical assays. In addition, we mapped a 50-amino acid region in LANA which was capable of abrogating the association of MCM6 with LANA and blocking DNA replication. We also detected LANA along with MCMs at the replication forks using a novel approach, isolation of proteins on nascent DNA (iPOND).


Assuntos
Antígenos Virais/genética , Replicação do DNA/genética , DNA Viral/genética , Fase G1/genética , Proteínas de Manutenção de Minicromossomo/genética , Proteínas Nucleares/genética , Fase S/genética , Replicação Viral/genética , Divisão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Genoma Viral/genética , Células HEK293 , Herpesvirus Humano 8/genética , Humanos , Origem de Replicação/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Sequências Repetidas Terminais/genética , Latência Viral/genética
17.
AIDS Res Hum Retroviruses ; 35(3): 348-356, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30565469

RESUMO

Female sex hormones, the octamer-binding transcription factor 4 (OCT4), and human endogenous retroviruses (HERVs) are all involved in the development of breast cancer. However, whether there are cross talks between these factors to promote breast cancer is still unknown. Using the T47D human breast cancer cell line, we have found that estradiol and progesterone synergistically activate HERV-K through nuclear receptors. The progesterone receptor (isoform B) binds a progesterone-response element (PRE) in a long terminal repeat (LTR5HS) of HERV-K. There is another transcription factor-binding element in the LTR, the octamer motif, which is required for the hormones to activate gene transcription downstream of the LTR. Gel shift assays and co-immunoprecipitation indicate that the progesterone receptor (PR) and the OCT4 transcription factor interact on the protein level. Methylation of the PRE enhances binding of the PR. These findings help to elucidate the previously unknown cross talks among the sex hormones, OCT4, and HERVs in contributing to breast cancer proliferation and tumorigenesis, which may be useful in guiding further development of cancer therapies.


Assuntos
Neoplasias da Mama/metabolismo , Retrovirus Endógenos/metabolismo , Estradiol/farmacologia , Fator 3 de Transcrição de Octâmero/metabolismo , Progesterona/farmacologia , Neoplasias da Mama/patologia , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA , Retrovirus Endógenos/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Receptores de Progesterona/metabolismo , Elementos de Resposta/genética , Sequências Repetidas Terminais/genética , Transcrição Genética , Transfecção
18.
RNA Biol ; 16(4): 404-412, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30252595

RESUMO

The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3' repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Francisella/genética , Sequências Repetidas Terminais/genética , Clivagem do DNA , DNA Intergênico/genética , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , RNA Guia/metabolismo , Fator Rho/metabolismo , Terminação da Transcrição Genética
19.
Plant Cell Rep ; 38(4): 455-458, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30465094

RESUMO

KEY MESSAGE: A successful example of transposon deletion via CRISPR/Cas9-mediated genome editing suggests a novel alternative approach to plant breeding. Transposition of transposable elements (TEs) can affect adjacent genes, leading to changes in genetic traits. Expression levels and patterns, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus can be affected. Artificial modification of loci adjacent to TEs, or TEs themselves, by genome editing could mimic the translocation of TEs that occurs in nature, suggesting that it might be possible to produce novel plants by modification of TEs via genome editing. To our knowledge, there are no reports thus far of modification of TEs by genome editing in plants. In this study, we performed targeted deletion of the Tos17 retrotransposon, which is flanked at both ends by long terminal repeat (LTR) sequences, via genome editing in rice. We succeeded in targeted mutagenesis of the LTR, and targeted deletion between LTRs, in calli transformed with CRISPR/Cas9 vectors for the Tos17 LTR. Moreover, we also successfully obtained regenerated plants derived from transformed calli and plants homozygous for lacking Tos17 in the next generation. Taken together, our results demonstrate successful deletion of the Tos17 retrotransposon from the rice genome by targeted mutagenesis using CRISPR/Cas9. We believe that this strategy could be applied to other TEs in many plant species, providing a rapid breeding technology as an alternative means to re-activate expression of agronomically important genes that have been inactivated by TE insertion, especially in plants such as fruit trees, in which it is difficult to maintain parental agronomical traits by cross-breeding due to high heterozygosity.


Assuntos
Oryza/genética , Retroelementos/genética , Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , Edição de Genes/métodos , Genoma de Planta/genética , Sequências Repetidas Terminais/genética
20.
Plant Physiol Biochem ; 135: 460-468, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30497974

RESUMO

Frequent somatic variations exist in pitaya (Hylocereus undatus) plants grown under abiotic stress conditions. Long terminal repeat (LTR) retrotransposons can be activated under stressful conditions and play key roles in plant genetic variation and evolution. However, whether LTR retrotransposons promotes pitaya somatic variations by regulating abiotic stress responses is still uncertain. In this study, transcriptionally active LTR retrotransposons were identified in pitaya after exposure to a number of stress factors, including in vitro culturing, osmotic changes, extreme temperatures and hormone treatments. In total, 26 LTR retrotransposon reverse transcriptase (RT) cDNA sequences were isolated and identified as belonging to 9 Ty1-copia and 4 Ty3-gypsy families. Several RT cDNA sequences had differing similarity levels with RTs from pitaya genomic DNA and other plant species, and were differentially expressed in pitaya under various stress conditions. LTR retrotransposons accounted for at least 13.07% of the pitaya genome. HuTy1P4 had a high copy number and low expression level in young stems of pitaya, and its expression level increased after exposure to hormones and abiotic stresses, including in vitro culturing, osmotic changes, cold and heat. HuTy1P4 may have been subjected to diverse transposon events in 13 pitaya plantlets successively subcultured for four cycles. Thus, the expression levels of these retrotransposons in pitaya were associated with stress responses and may be involved in the occurrence of the somaclonal variation in pitaya.


Assuntos
Cactaceae/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Retroelementos/genética , Sequências Repetidas Terminais/genética , Cactaceae/fisiologia , Clonagem Molecular , DNA de Plantas/genética , Genoma de Planta/genética , Genoma de Planta/fisiologia , Retroelementos/fisiologia , Análise de Sequência de DNA , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Sequências Repetidas Terminais/fisiologia
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