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1.
EMBO J ; 34(2): 169-83, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25452498

RESUMO

The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria.


Assuntos
Bacillus subtilis/virologia , Bacteriófagos/genética , Bacteriófagos/patogenicidade , Metilação de DNA , Metilases de Modificação do DNA/genética , Genoma Microbiano , Virulência/genética , Bacillus subtilis/genética , Bacteriófagos/crescimento & desenvolvimento , Evolução Biológica , Metilases de Modificação do DNA/metabolismo , DNA Bacteriano/genética , DNA Viral/genética , Filogenia
2.
Nucleic Acids Res ; 45(11): e95, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28335028

RESUMO

Cells regulate biological responses in part through changes in transcription start sites (TSS) or cleavage and polyadenylation sites (PAS). To fully understand gene regulatory networks, it is therefore critical to accurately annotate cell type-specific TSS and PAS. Here we present a simple and straightforward approach for genome-wide annotation of 5΄- and 3΄-RNA ends. Our approach reliably discerns bona fide PAS from false PAS that arise due to internal poly(A) tracts, a common problem with current PAS annotation methods. We applied our methodology to study the impact of temperature on the Drosophila melanogaster head transcriptome. We found hundreds of previously unidentified TSS and PAS which revealed two interesting phenomena: first, genes with multiple PASs tend to harbor a motif near the most proximal PAS, which likely represents a new cleavage and polyadenylation signal. Second, motif analysis of promoters of genes affected by temperature suggested that boundary element association factor of 32 kDa (BEAF-32) and DREF mediates a transcriptional program at warm temperatures, a result we validated in a fly line where beaf-32 is downregulated. These results demonstrate the utility of a high-throughput platform for complete experimental and computational analysis of mRNA-ends to improve gene annotation.


Assuntos
Drosophila melanogaster/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Região 3'-Flanqueadora , Região 5'-Flanqueadora , Animais , Sequência de Bases , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Exonucleases/química , Genes de Insetos , Anotação de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease H/química , Transcriptoma
3.
Nucleic Acids Res ; 45(16): e148, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28934479

RESUMO

The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described 'naive-like' memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/química , Receptores de Antígenos de Linfócitos T/química , Análise de Sequência de RNA/métodos , Software , Algoritmos , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Célula Única , Vírus da Febre Amarela/imunologia
4.
Life Sci Alliance ; 2(4)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31451449

RESUMO

RNA sequencing of single B cells provides simultaneous measurements of the cell state and its antigen specificity as determined by the B-cell receptor (BCR). However, to uncover the latter, further reconstruction of the BCR sequence is needed. We present BRAPeS ("BCR Reconstruction Algorithm for Paired-end Single cells" ), an algorithm for reconstructing BCRs from short-read paired-end single-cell RNA sequencing. BRAPeS is accurate and achieves a high success rate even at very short (25 bp) read length, which can decrease the cost and increase the number of cells that can be analyzed compared with long reads. BRAPeS is publicly available at the following link: https://github.com/YosefLab/BRAPeS.


Assuntos
Receptores de Antígenos de Linfócitos B/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Algoritmos , Linfócitos B/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sequenciamento do Exoma
5.
Cell Syst ; 6(3): 381-394.e7, 2018 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-29454939

RESUMO

Most well-characterized enhancers are deeply conserved. In contrast, genome-wide comparative studies of steady-state systems showed that only a small fraction of active enhancers are conserved. To better understand conservation of enhancer activity, we used a comparative genomics approach that integrates temporal expression and epigenetic profiles in an innate immune system. We found that gene expression programs diverge among mildly induced genes, while being highly conserved for strongly induced genes. The fraction of conserved enhancers varies greatly across gene expression programs, with induced genes and early-response genes, in particular, being regulated by a higher fraction of conserved enhancers. Clustering of conserved accessible DNA sequences within enhancers resulted in over 60 sequence motifs including motifs for known factors, as well as many with unknown function. We further show that the number of instances of these motifs is a strong predictor of the responsiveness of a gene to pathogen detection.


Assuntos
Elementos Facilitadores Genéticos/genética , Genômica/métodos , Imunidade Inata/genética , Animais , Sequência Conservada/genética , Epigênese Genética/genética , Evolução Molecular , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
6.
Cell Rep ; 11(2): 308-20, 2015 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-25843718

RESUMO

Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5' RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes.


Assuntos
Diferenciação Celular/genética , Impressão Genômica/genética , Células-Tronco Pluripotentes Induzidas , Transcrição Gênica , Metilação de DNA/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética
7.
Nat Commun ; 6: 7056, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25952406

RESUMO

The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. Here we show that Clk mRNA is strongly regulated post-transcriptionally through its 3' UTR. Flies expressing Clk transgenes without normal 3' UTR exhibit variable CLK-driven transcription and circadian behaviour as well as ectopic expression of CLK-target genes in the brain. In these flies, the number of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. Moreover, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall our results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila.


Assuntos
Proteínas CLOCK/metabolismo , Ritmo Circadiano/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica , Transcrição Gênica , Regiões 3' não Traduzidas/genética , Animais , Proteínas Argonautas/metabolismo , Comportamento Animal , Sítios de Ligação , Relógios Biológicos/genética , Encéfalo/metabolismo , Proteínas CLOCK/genética , Proteínas de Drosophila/genética , Retroalimentação Fisiológica , Imunofluorescência , MicroRNAs/metabolismo , Modelos Biológicos , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência , Processos Estocásticos , Fatores de Tempo
8.
Diabetes ; 64(9): 3172-81, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25931473

RESUMO

Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic α- and ß-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase ß-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, ß-, and δ-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult α- and ß-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal ß-cells. In addition, within highly purified adult glucagon-expressing α-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet α- and ß-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.


Assuntos
Feto/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , RNA/genética , Células Secretoras de Somatostatina/metabolismo , Adolescente , Adulto , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Humanos , Ilhotas Pancreáticas/citologia , Masculino , Pessoa de Meia-Idade , Gravidez , Segundo Trimestre da Gravidez , Análise de Sequência de RNA , Adulto Jovem
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