RESUMO
Changes in transcription factor binding sites (TFBSs) can alter the spatiotemporal expression pattern and transcript abundance of genes. Loss and gain of TFBSs were shown to cause shifts in expression patterns in numerous cases. However, we know little about the evolution of extended regulatory sequences incorporating many TFBSs. We compare, across the crucifers (Brassicaceae, cabbage family), the sequences between the translated regions of Arabidopsis Bsister (ABS)-like MADS-box genes (including paralogous GOA-like genes) and the next gene upstream, as an example of family-wide evolution of putative upstream regulatory regions (PURRs). ABS-like genes are essential for integument development of ovules and endothelium formation in seeds of Arabidopsis thaliana. A combination of motif-based gene ontology enrichment and reporter gene analysis using A. thaliana as common trans-regulatory environment allows analysis of selected Brassicaceae Bsister gene PURRs. Comparison of TFBS of transcriptionally active ABS-like genes with those of transcriptionally largely inactive GOA-like genes shows that the number of in silico predicted TFBS) is similar between paralogs, emphasizing the importance of experimental verification for in silico characterization of TFBS activity and analysis of their evolution. Further, our data show highly conserved expression of Brassicaceae ABS-like genes almost exclusively in the chalazal region of ovules. The Arabidopsis-specific insertion of a transposable element (TE) into the ABS PURRs is required for stabilizing this spatially restricted expression, while other Brassicaceae achieve chalaza-specific expression without TE insertion. We hypothesize that the chalaza-specific expression of ABS is regulated by cis-regulatory elements provided by the TE.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Brassica , Brassicaceae , Arabidopsis/metabolismo , Brassicaceae/genética , Brassicaceae/metabolismo , Elementos de DNA Transponíveis , Proteínas de Arabidopsis/genética , Sementes/genética , Brassica/genética , Regulação da Expressão Gênica de PlantasRESUMO
Rice blast is a very serious disease caused by Magnaporthe oryzae, which threatens rice production and food supply throughout the world. The avirulence (AVR) genes of rice blast are perceived by the corresponding rice blast resistance (R) genes and prompt specific resistance. A mutation in AVR is a major force for new virulence. Exploring mutations in AVR among M. oryzae isolates from rice production fields could aid assessment of the efficacy and durability of R genes. We studied the probable molecular-evolutionary patterns of AVR-Pib alleles by assaying their DNA-sequence diversification and examining their avirulence to the corresponding Pib resistance gene under natural conditions in the extremely genetically diverse of rice resources of Yunnan, China. PCRs detected results from M. oryzae genomic DNA and revealed that 162 out of 366 isolates collected from Yunnan Province contained AVR-Pib alleles. Among them, 36.1-73.3% isolates from six different rice production areas of Yunnan contained AVR-Pib alleles. Furthermore, 36 (28.6%) out of 126 isolates had a transposable element (TE) insertion in AVR-Pib, which resulted in altered virulence. The TE insertion was identified in isolates from rice rather than from Musa nana Lour. Twelve AVR-Pib haplotypes encoding three novel AVR-Pib variants were identified among the remaining 90 isolates. AVR-Pib alleles evolved to virulent forms from avirulent forms by base substitution and TE insertion of Pot2 and Pot3 in the 5' untranslated region of AVR-Pib. These findings support the hypothesis that functional AVR-Pib possesses varied sequence structures and can escape surveillance by hosts via multiple variation manners.
Assuntos
Magnaporthe , Oryza , Elementos de DNA Transponíveis/genética , Variação Genética , Magnaporthe/genética , China , Oryza/genética , Doenças das Plantas/genéticaRESUMO
MutS homolog 1 (MSH1) is involved in the recombining and repairing of organelle genomes and is essential for maintaining their stability. Previous studies indicated that the length of the gene varied greatly among species and detected species-specific partial gene duplications in Physcomitrella patens. However, there are critical gaps in the understanding of the gene size expansion, and the extent of the partial gene duplication of MSH1 remains unclear. Here, we screened MSH1 genes in 85 selected species with genome sequences representing the main clades of green plants (Viridiplantae). We identified the MSH1 gene in all lineages of green plants, except for nine incomplete species, for bioinformatics analysis. The gene is a singleton gene in most of the selected species with conserved amino acids and protein domains. Gene length varies greatly among the species, ranging from 3234 bp in Ostreococcus tauri to 805,861 bp in Cycas panzhihuaensis. The expansion of MSH1 repeatedly occurred in multiple clades, especially in Gymnosperms, Orchidaceae, and Chloranthus spicatus. MSH1 has exceptionally long introns in certain species due to the gene length expansion, and the longest intron even reaches 101,025 bp. And the gene length is positively correlated with the proportion of the transposable elements (TEs) in the introns. In addition, gene structure analysis indicated that the MSH1 of green plants had undergone parallel intron gains and losses in all major lineages. However, the intron number of seed plants (gymnosperm and angiosperm) is relatively stable. All the selected gymnosperms contain 22 introns except for Gnetum montanum and Welwitschia mirabilis, while all the selected angiosperm species preserve 21 introns except for the ANA grade. Notably, the coding region of MSH1 in algae presents an exceptionally high GC content (47.7% to 75.5%). Moreover, over one-third of the selected species contain species-specific partial gene duplications of MSH1, except for the conserved mosses-specific partial gene duplication. Additionally, we found conserved alternatively spliced MSH1 transcripts in five species. The study of MSH1 sheds light on the evolution of the long genes of green plants.
Assuntos
Magnoliopsida , Viridiplantae , Íntrons/genética , Duplicação Gênica , Processamento Alternativo , Biologia Computacional , Cycadopsida , Proteínas MutSRESUMO
BACKGROUND: Transposable elements (TE) account for more than 50% of human genome. It has been reported that some types of TEs are dynamically regulated in the reprogramming of human cell lines. However, it is largely unknown whether some TEs in Macaca mulatta are also regulated during the reprogramming of cell lines of monkey. RESULTS: Here, we systematically examined the transcriptional activities of TEs during the conversion of Macaca mulatta fibroblast cells to neuroepithelial stem cells (NESCs). Hundreds of TEs were dynamically regulated during the reprogramming of Macaca mulatta fibroblast cells. Furthermore, 48 Long Terminal Repeats (LTRs), as well as some integrase elements, of Macaca endogenous retrovirus 3 (MacERV3) were transiently activated during the early stages of the conversion process, some of which were further confirmed with PCR experiments. These LTRs were potentially bound by critical transcription factors for reprogramming, such as KLF4 and ETV5. CONCLUSION: These results suggest that the transcription of TEs are delicately regulated during the reprogramming of Macaca mulatta fibroblast cells. Although the family of ERVs activated during the reprogramming of fibroblast cells in Macaca mulatta is different from those in the reprogramming of human fibroblast cells, our results suggest that the activation of some ERVs is a conserved mechanism in primates for converting fibroblast cells to stem cells.
Assuntos
Elementos de DNA Transponíveis , Sequências Repetidas Terminais , Animais , Elementos de DNA Transponíveis/genética , Fibroblastos , Humanos , Fator 4 Semelhante a Kruppel , Macaca mulatta , Células-TroncoRESUMO
Innovations in high-throughout sequencing approaches are being marshaled to both reveal the composition of the abundant and heterogeneous noncoding RNAs that populate cell nuclei and lend insight to the mechanisms by which noncoding RNAs influence chromosome biology and gene expression. This review focuses on some of the recent technological developments that have enabled the isolation of nascent transcripts and chromatin-associated and DNA-interacting RNAs. Coupled with emerging genome assembly and analytical approaches, the field is poised to achieve a comprehensive catalog of nuclear noncoding RNAs, including those derived from repetitive regions within eukaryotic genomes. Herein, particular attention is paid to the challenges and advances in the sequence analyses of repeat and transposable element-derived noncoding RNAs and in ascribing specific function(s) to such RNAs.
Assuntos
RNA não Traduzido , Sequências Repetitivas de Ácido Nucleico , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interferência de RNA , TranscriptomaRESUMO
The reverse transcriptases (RTs) encoded by mobile group II introns and other non-LTR retroelements differ from retroviral RTs in being able to template-switch efficiently from the 5' end of one template to the 3' end of another with little or no complementarity between the donor and acceptor templates. Here, to establish a complete kinetic framework for the reaction and to identify conditions that more efficiently capture acceptor RNAs or DNAs, we used a thermostable group II intron RT (TGIRT; GsI-IIC RT) that can template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end. We found that the rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for nontemplated nucleotide addition of a complementary nucleotide to the 3' end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decreased the template-switching rate, even when complementary to the 3' end of the acceptor template. The reliance on only a single bp with the 3' nucleotide of the acceptor together with discrimination against mismatches and the high processivity of group II intron RTs enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3' end. We discuss the possible biological functions of the template-switching activity of group II intron- and other non-LTR retroelement-encoded RTs, as well as the optimization of this activity for adapter addition in RNA- and DNA-Seq protocols.
Assuntos
Íntrons , Nucleotídeos/genética , DNA Polimerase Dirigida por RNA/metabolismo , RNA-Seq/métodos , Retroelementos/genética , Moldes Genéticos , Animais , Primers do DNA , Elementos de DNA Transponíveis , Teste de Complementação Genética , Insetos , Cinética , RNA/genética , Retroviridae/genética , Temperatura , Sequenciamento do ExomaRESUMO
The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) after aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway or, alternatively, for enhancing homology-directed repair to facilitate the generation of a specific mutation (or "knock-in"). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA activity. Reliable methods for enriching and identifying desired mutants are also lacking. Here we describe a method using the Piggybac transposon for stable genomic integration of an H2B-GFP reporter or a hygromycin resistance gene for assaying Cas9 target cleavage and homology-directed repair. The H2B-GFP fusion protein provides increased stability and an obvious pattern of nuclear localization. This method, called SRIRACCHA (i.e. a stable, but reversible, integrated reporter for assaying CRISPR/Cas-stimulated HDR activity), enables the enrichment of mutants via selection of GFP-positive or hygromycin-resistant mammalian cells (immortalized or non-immortalized) as a surrogate for the modification of the endogenous target site. Currently available hyperactive Piggybac transposase mutants allow both delivery and removal of the surrogate reporters, with minimal risk of generating undesirable mutations. This assay permits rapid screening for efficient guide RNAs and the accelerated identification of mutant clones and is applicable to many cell types. We foresee the utility of this approach in contexts in which the maintenance of genomic integrity is essential, for example, when engineering cells for therapeutic purposes.
Assuntos
Sistemas CRISPR-Cas , Deleção de Genes , Marcação de Genes/métodos , Vetores Genéticos/genética , Animais , Linhagem Celular Tumoral , CamundongosRESUMO
The evolutionary dynamics of the conflict between transposable elements (TEs) and their host genome remain elusive. This conflict will be intense in stress-adapted plants as stress can often reactivate TEs. Mangroves reduce TE load convergently in their adaptation to intertidal environments and thus provide a unique opportunity to address the host-TE conflict and its interaction with stress adaptation. Using the mangrove Rhizophora apiculata as a model, we investigated methylation and short interfering RNA (siRNA) targeting patterns in relation to the abundance and age of long terminal repeat (LTR) retrotransposons. We also examined the distance of LTR retrotransposons to genes, the impact on neighboring gene expression and population frequencies. We found differential accumulation amongst classes of LTR retrotransposons despite high overall methylation levels. This can be attributed to 24-nucleotide siRNA-mediated CHH methylation preferentially targeting Gypsy elements, particularly in their LTR regions. Old Gypsy elements possess unusually abundant siRNAs which show cross-mapping to young copies. Gypsy elements appear to be closer to genes and under stronger purifying selection than other classes. Our results suggest a continuous host-TE battle masked by the TE load reduction in R. apiculata. This conflict may enable mangroves, such as R. apiculata, to maintain genetic diversity and thus evolutionary potential during stress adaptation.
Assuntos
Adaptação Fisiológica/genética , Avicennia/genética , Avicennia/fisiologia , Metilação de DNA/genética , Retroelementos/genética , Estresse Fisiológico/genética , Sequências Repetidas Terminais/genética , Sequência de Bases , Evolução Molecular , Dosagem de Genes , Regulação da Expressão Gênica de Plantas , RNA Interferente Pequeno/metabolismoRESUMO
Retrotransposons are eukaryotic mobile genetic elements that transpose by reverse transcription of an RNA intermediate and are derived from retroviruses. The Ty1 retrotransposon of Saccharomyces cerevisiae belongs to the Ty1/Copia superfamily, which is present in every eukaryotic genome. Insertion of Ty1 elements into the S. cerevisiae genome, which occurs upstream of genes transcribed by RNA Pol III, requires the Ty1 element-encoded integrase (IN) protein. Here, we report that Ty1-IN interacts in vivo and in vitro with RNA Pol III-specific subunits to mediate insertion of Ty1 elements upstream of Pol III-transcribed genes. Purification of Ty1-IN from yeast cells followed by mass spectrometry (MS) analysis identified an enrichment of peptides corresponding to the Rpc82/34/31 and Rpc53/37 Pol III-specific subcomplexes. GFP-Trap purification of multiple GFP-tagged RNA Pol III subunits from yeast extracts revealed that the majority of Pol III subunits co-purify with Ty1-IN but not two other complexes required for Pol III transcription, transcription initiation factors (TF) IIIB and IIIC. In vitro binding studies with bacterially purified RNA Pol III proteins demonstrate that Rpc31, Rpc34, and Rpc53 interact directly with Ty1-IN. Deletion of the N-terminal 280 amino acids of Rpc53 abrogates insertion of Ty1 elements upstream of the hot spot SUF16 tRNA locus and abolishes the interaction of Ty1-IN with Rpc37. The Rpc53/37 complex therefore has an important role in targeting Ty1-IN to insert Ty1 elements upstream of Pol III-transcribed genes.
Assuntos
Integrases/fisiologia , RNA Polimerase III/metabolismo , Retroelementos , Saccharomyces cerevisiae/genética , Integrases/química , Mutagênese Insercional , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , RNA Polimerase III/química , RNA Polimerase III/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
The prion protein (PrP) seems to exert both neuroprotective and neurotoxic activities. The toxic activities are associated with the C-terminal globular parts in the absence of the flexible N terminus, specifically the hydrophobic domain (HD) or the central region (CR). The wild type prion protein (PrP-WT), having an intact flexible part, exhibits neuroprotective qualities by virtue of diminishing many of the cytotoxic effects of these mutant prion proteins (PrPΔHD and PrPΔCR) when coexpressed. The prion protein family member Doppel, which possesses a three-dimensional fold similar to the C-terminal part of PrP, is also harmful to neuronal and other cells in various models, a phenotype that can also be eliminated by the coexpression of PrP-WT. In contrast, another prion protein family member, Shadoo (Sho), a natively disordered protein possessing structural features similar to the flexible N-terminal tail of PrP, exhibits PrP-WT-like protective properties. Here, we report that, contrary to expectations, Sho expression in SH-SY5Y or HEK293 cells induces the same toxic phenotype of drug hypersensitivity as PrPΔCR. This effect is exhibited in a dose-dependent manner and is also counteracted by the coexpression of PrP-WT. The opposing effects of Shadoo in different model systems revealed here may be explored to help discern the relationship of the various toxic activities of mutant PrPs with each other and the neurotoxic effects seen in neurodegenerative diseases, such as transmissible spongiform encephalopathy and Alzheimer disease.
Assuntos
Resistência a Múltiplos Medicamentos , Hepatócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Príons/metabolismo , Animais , Anti-Infecciosos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas Ligadas por GPI , Deleção de Genes , Células HEK293 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos Mutantes , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Priônicas , Príons/química , Príons/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction.
Assuntos
Catelicidinas/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Deleção de Genes , Lipopolissacarídeos/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Catelicidinas/química , Membrana Celular/química , Membrana Celular/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Expressão Gênica , Heptoses/química , Heptoses/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Lipopolissacarídeos/química , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Eletricidade EstáticaRESUMO
The retrotransposon DIRS-1 is the most abundant retroelement in Dictyostelium discoideum and constitutes the pericentromeric heterochromatin of the six chromosomes in D. discoideum. The vast majority of cellular siRNAs is derived from DIRS-1, suggesting that the element is controlled by RNAi-related mechanisms. We investigated the role of two of the five Argonaute proteins of D. discoideum, AgnA and AgnB, in DIRS-1 silencing. Deletion of agnA resulted in the accumulation of DIRS-1 transcripts, the expression of DIRS-1-encoded proteins, and the loss of most DIRS-1-derived secondary siRNAs. Simultaneously, extrachromosomal single-stranded DIRS-1 DNA accumulated in the cytoplasm of agnA- strains. These DNA molecules appear to be products of reverse transcription and thus could represent intermediate structures before transposition. We further show that transitivity of endogenous siRNAs is impaired in agnA- strains. The deletion of agnB alone had no strong effect on DIRS-1 transposon regulation. However, in agnA-/agnB- double mutant strains strongly reduced accumulation of extrachromosomal DNA compared with the single agnA- strains was observed.
Assuntos
Proteínas Argonautas/genética , DNA de Protozoário/genética , Dictyostelium/genética , Proteínas de Protozoários/genética , RNA Interferente Pequeno/genética , Retroelementos/genética , Proteínas Argonautas/metabolismo , Western Blotting , DNA de Protozoário/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Dictyostelium/metabolismo , Deleção de Genes , Expressão Gênica , Mutação , Proteínas de Protozoários/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transformação GenéticaRESUMO
PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3'-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs.
Assuntos
Células Intersticiais de Cajal/metabolismo , Intestino Delgado/metabolismo , Estágio Paquíteno/genética , RNA Interferente Pequeno/genética , Testículo/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Regulação da Expressão Gênica , Intestino Delgado/citologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Células de Sertoli/metabolismo , Espermátides/metabolismo , Espermatócitos/metabolismo , Testículo/citologia , TranscriptomaRESUMO
The development of age-associated diseases is related to the accumulation of senescent cells in the body. These are old non-functional cells with impaired metabolism, which are unable to divide. Such cells are also resistant to programmed cell death and prone to spontaneous production of some inflammatory factors. The accumulation of senescent cells is related to the age-associated dysfunction of organs and tissues as well as chronic inflammation that enhances with age. In the young organism, senescent cells are removed with the innate immunity system. However, the efficiency of this process decreases with age. Nowadays, more and more evidences are accumulating to support the involvement of specific immunity and T-lymphocytes in the fight against senescent cells. It has great physiological importance since the efficient elimination of senescent cells requires a high diversity of antigen-recognizing receptors to cover the entire spectrum of senescent-associated antigens with high precision and specificity. Developing the approaches of T-cell immunity stimulation to generate or amplify a physiological immune response against senescent cells can provide new perspectives to extend active longevity. In this mini-review, the authors summarize the current understanding of the role of T-cell immunity in the fight against senescent cells and discuss the prospects of stimulating adaptive immunity for combating the accumulation of senescent cells that occurs with age.
Assuntos
Senescência Celular , Linfócitos T , Longevidade , ApoptoseRESUMO
Backgrounds: Hepatitis B virus (HBV) infection is a major risk factor for chronic liver diseases and liver cancer (mainly hepatocellular carcinoma, HCC), while the underlying mechanisms and host-virus interactions are still largely elusive. Methods: We applied HiC sequencing to HepG2 (HBV-) and HepG2-2.2.15 (HBV+) cell lines and combined them with public HCC single-cell RNA-seq data, HCC bulk RNA-seq data, and both genomic and epigenomic ChIP-seq data to reveal potential disease mechanisms of HBV infection and host-virus interactions reflected by 3D genome organization. Results: We found that HBV enhanced overall proximal chromatin interactions (CIs) of liver cells and primarily affected regional CIs on chromosomes 13, 14, 17, and 22. Interestingly, HBV altered the boundaries of many topologically associating domains (TADs), and genes nearby these boundaries showed functional enrichment in cell adhesion which may promote cancer metastasis. Moreover, A/B compartment analysis revealed dramatic changes on chromosomes 9, 13 and 21, with more B compartments (inactive or closed) shifting to A compartments (active or open). The A-to-B regions (closing) harbored enhancers enriched in the regulation of inflammatory responses, whereas B-to-A regions (opening) were enriched for transposable elements (TE). Furthermore, we identified large HBV-induced structural variations (SVs) that disrupted tumor suppressors, NLGN4Y and PROS1. Finally, we examined differentially expressed genes and TEs in single hepatocytes with or without HBV infection, by using single-cell RNA-seq data. Consistent with our HiC sequencing findings, two upregulated genes that promote HBV replication, HNF4A and NR5A2, were located in regions with HBV-enhanced CIs, and five TEs were located in HBV-activated regions. Therefore, HBV may promote liver diseases by affecting the human 3D genome structure. Conclusion: Our work promotes mechanistic understanding of HBV infection and host-virus interactions related to liver diseases that affect billions of people worldwide. Our findings may also have implications for novel immunotherapeutic strategies targeting HBV infection.
Assuntos
Carcinoma Hepatocelular , Hepatite B , Neoplasias Hepáticas , Humanos , Vírus da Hepatite B/fisiologia , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/patologia , Interações entre Hospedeiro e Microrganismos , Hepatite B/complicaçõesRESUMO
Angiosperms form the largest phylum within the Plantae kingdom and show remarkable genetic variation due to the considerable difference in the nuclear genome size of each species. Transposable elements (TEs), mobile DNA sequences that can amplify and change their chromosome position, account for much of the difference in nuclear genome size between individual angiosperm species. Considering the dramatic consequences of TE movement, including the complete loss of gene function, it is unsurprising that the angiosperms have developed elegant molecular strategies to control TE amplification and movement. Specifically, the RNA-directed DNA methylation (RdDM) pathway, directed by the repeat-associated small-interfering RNA (rasiRNA) class of small regulatory RNA, forms the primary line of defense to control TE activity in the angiosperms. However, the miniature inverted-repeat transposable element (MITE) species of TE has at times avoided the repressive effects imposed by the rasiRNA-directed RdDM pathway. MITE proliferation in angiosperm nuclear genomes is due to their preference to transpose within gene-rich regions, a pattern of transposition that has enabled MITEs to gain further transcriptional activity. The sequence-based properties of a MITE results in the synthesis of a noncoding RNA (ncRNA), which, after transcription, folds to form a structure that closely resembles those of the precursor transcripts of the microRNA (miRNA) class of small regulatory RNA. This shared folding structure results in a MITE-derived miRNA being processed from the MITE-transcribed ncRNA, and post-maturation, the MITE-derived miRNA can be used by the core protein machinery of the miRNA pathway to regulate the expression of protein-coding genes that harbor homologous MITE insertions. Here, we outline the considerable contribution that the MITE species of TE have made to expanding the miRNA repertoire of the angiosperms.
RESUMO
Transposable elements (TEs) are responsible for significant genomic variation in plants. Our understanding of the evolutionary forces shaping TE polymorphism has lagged behind other mutations because of the difficulty of accurately identifying TE polymorphism in short-read population genomic data. However, new approaches allow us to quantify TE polymorphisms in population datasets and address fundamental questions about the evolution of these polymorphisms. Here, we discuss how insertional biases shape where, when, and how often TEs insert throughout the genome. Next, we examine mechanisms by which TEs can affect phenotype. Finally, we evaluate current evidence for selection on TE polymorphisms. All together, it is clear that TEs are important, but underappreciated, contributors to intraspecific phenotypic variation, and that understanding the dynamics governing TE polymorphism is crucial for evolutionary biologists interested in the maintenance of variation.
Assuntos
Elementos de DNA Transponíveis , Evolução Molecular , Variação Biológica da População , Elementos de DNA Transponíveis/genética , GenômicaRESUMO
Transposable elements (TEs) are mobile genetic elements that can randomly integrate into other genomic sites. They have successfully replicated and now occupy around 40% of the total DNA sequence in humans. TEs in the genome have a complex relationship with the host cell, being both potentially deleterious and advantageous at the same time. Only a tiny minority of TEs are still capable of transposition, yet their fossilized sequence fragments are thought to be involved in various molecular processes, such as gene transcriptional activity, RNA stability and subcellular localization, and chromosomal architecture. TEs have also been implicated in biological processes, although it is often hard to reveal cause from correlation due to formidable technical issues in analyzing TEs. In this review, we compare and contrast two views of TE activity: one in the pluripotent state, where TEs are broadly beneficial, or at least mechanistically useful, and a second state in human disease, where TEs are uniformly considered harmful.
RESUMO
Transposable elements (TEs) are mobile genetic elements that made up about half the human genome. Among them, the autonomous non-LTR retrotransposon long interspersed nuclear element-1 (L1) is the only currently active TE in mammals and covers about 17% of the mammalian genome. L1s exert their function as structural elements in the genome, as transcribed RNAs to influence chromatin structure and as retrotransposed elements to shape genomic variation in somatic cells. L1s activity has been shown altered in several diseases of the nervous system. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expansion of a CAG repeat in the HTT gene which leads to a gradual loss of neurons most prominently in the striatum and, to a lesser extent, in cortical brain regions. The length of the expanded CAG tract is related to age at disease onset, with longer repeats leading to earlier onset. Here we carried out bioinformatic analysis of public RNA-seq data of a panel of HD mouse models showing that a decrease of L1 RNA expression recapitulates two hallmarks of the disease: it correlates to CAG repeat length and it occurs in the striatum, the site of neurodegeneration. Results were then experimentally validated in Htt Q111 knock-in mice. The expression of L1-encoded proteins was independent from L1 RNA levels and differentially regulated in time and tissues. The pattern of expression L1 RNAs in human HD post-mortem brains showed similarity to mouse models of the disease. This work suggests the need for further study of L1s in HD and adds support to the current hypothesis that dysregulation of TEs may be involved in neurodegenerative diseases.
RESUMO
Human endogenous retroviruses (HERV), ancient integrations of exogenous viruses, make up 8% of our genome. Long thought of as mere vestigial genetic elements, evidence is now accumulating to suggest a potential functional role in numerous pathologies including neurodegenerative diseases, autoimmune disorders, and multiple cancers. The youngest member of this group of transposable elements is HERV-K (HML-2). Like the majority of HERV sequences, significant post-insertional mutations have disarmed HERV-K (HML-2), preventing it from producing infectious viral particles. However, some insertions have retained limited coding capacity, and complete open reading frames for all its constituent proteins can be found throughout the genome. For this reason HERV-K (HML-2) has garnered more attention than its peers. The tight epigenetic control thought to suppress expression in healthy tissue is lost during carcinogenesis. Upregulation of HERV-K (HML-2) derived mRNA and protein has been reported in a variety of solid and liquid tumour types, and while causality has yet to be established, progressively more data are emerging to suggest this phenomenon may contribute to tumour growth and metastatic capacity. Herein we discuss its potential utility as a diagnostic tool and therapeutic target in light of the current in vitro, in vivo and clinical evidence linking HERV-K (HML-2) to tumour progression.