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1.
Hum Mutat ; 40(7): 865-878, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31026367

RESUMO

Mendelian diseases have shown to be an and efficient model for connecting genotypes to phenotypes and for elucidating the function of genes. Whole-exome sequencing (WES) accelerated the study of rare Mendelian diseases in families, allowing for directly pinpointing rare causal mutations in genic regions without the need for linkage analysis. However, the low diagnostic rates of 20-30% reported for multiple WES disease studies point to the need for improved variant pathogenicity classification and causal variant prioritization methods. Here, we present the exome Disease Variant Analysis (eDiVA; http://ediva.crg.eu), an automated computational framework for identification of causal genetic variants (coding/splicing single-nucleotide variants and small insertions and deletions) for rare diseases using WES of families or parent-child trios. eDiVA combines next-generation sequencing data analysis, comprehensive functional annotation, and causal variant prioritization optimized for familial genetic disease studies. eDiVA features a machine learning-based variant pathogenicity predictor combining various genomic and evolutionary signatures. Clinical information, such as disease phenotype or mode of inheritance, is incorporated to improve the precision of the prioritization algorithm. Benchmarking against state-of-the-art competitors demonstrates that eDiVA consistently performed as a good or better than existing approach in terms of detection rate and precision. Moreover, we applied eDiVA to several familial disease cases to demonstrate its clinical applicability.

2.
Science ; 364(6436)2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30975860

RESUMO

To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress-related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.


Assuntos
Adaptação Fisiológica , Astronautas , Voo Espacial , Imunidade Adaptativa , Peso Corporal , Artérias Carótidas/diagnóstico por imagem , Espessura Intima-Media Carotídea , Dano ao DNA , Metilação de DNA , Microbioma Gastrointestinal , Instabilidade Genômica , Humanos , Masculino , Homeostase do Telômero , Fatores de Tempo , Estados Unidos , United States National Aeronautics and Space Administration
3.
Nat Commun ; 10(1): 579, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718479

RESUMO

The DNA base modification N6-methyladenine (m6A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method to detect base modifications. Here, we show that a neural network can improve m6A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.


Assuntos
Adenosina/análogos & derivados , Metilação de DNA/fisiologia , Análise de Sequência de DNA/métodos , Adenosina/análise , Algoritmos , Metilação de DNA/genética , Imunoprecipitação , Software
4.
Genome Res ; 2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30523036

RESUMO

Emerging Linked-Read technologies (aka Read-Cloud or barcoded short-reads) have revived interest in short-read technology as a viable way to understand large-scale structure in genomes and metagenomes. Linked-Read technologies, such as the 10x Chromium system, use a microfluidic system and a specialized set of 3' barcodes (aka UIDs) to tag short DNA reads sourced from the same long fragment of DNA; subsequently, the tagged reads are sequenced on standard short read platforms. This approach results in interesting compromises. Each long fragment of DNA is only sparsely covered by reads, no information about the ordering of reads from the same fragment is preserved, and 3' barcodes match reads from roughly 2-20 long fragments of DNA. However, compared to long read technologies the cost per base to sequence is far lower, far less input DNA is required, and their base error rate is that of Illumina short-reads.In this paper, we formally describe a particular algorithmic issue common to across Linked-Read technology: the deconvolution of reads with a single 3' barcode into clusters that represent single long fragments of DNA. We introduce Minerva, A graph-based algorithm that approximately solves the barcode deconvolution problem for metagenomic data (where reference genomes may be incomplete or unavailable). Additionally, we develop two demonstrations where the deconvolution of barcoded reads improves downstream results: improving the specificity of taxonomic assignments and improving the specificity of k-mer based clustering. To the best of our knowledge, we are the first to address the problem of barcode deconvolution in metagenomics.

5.
Front Immunol ; 9: 2397, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30386343

RESUMO

LRBA deficiency was first described in 2012 as an autosomal recessive disorder caused by biallelic mutations in the LRBA gene (OMIM #614700). It was initially characterized as producing early-onset hypogammaglobulinemia, autoimmune manifestations, susceptibility to inflammatory bowel disease, and recurrent infection. However, further reports expanded this phenotype (including patients without hypogammaglobulinemia) and described LRBA deficiency as a clinically variable syndrome with a wide spectrum of clinical manifestations. We present the case of a female patient who presented with type 1 diabetes, psoriasis, oral thrush, and enlarged liver and spleen at the age of 8 months. She later experienced recurrent bacterial and viral infections, including pneumococcal meningitis and Epstein Barr viremia. She underwent two consecutive stem cell transplants at the age of 8 and 9 years, and ultimately died. Samples from the patient and her parents were subjected to whole exome sequencing, which revealed a homozygous 1-bp insertion in exon 23 of the patient's LRBA gene, resulting in frameshift and premature stop codon. The patient's healthy mother was heterozygous for the mutation and her father tested wild-type. This finding suggested that either one copy of the paternal chromosome 4 bore a deletion including the LRBA locus, or the patient inherited two copies of the mutant maternal LRBA allele. The patient's sequencing data showed a 1-Mb loss of heterozygosity region in chromosome 4, including the LRBA gene. Comparative genomic hybridization array of the patient's and father's genomic DNA yielded normal findings, ruling out genomic copy number abnormalities. Here, we present the first case of LRBA deficiency due to a uniparental disomy (UPD). In contrast to classical Mendelian inheritance, UPD involves inheritance of 2 copies of a chromosomal region from only 1 parent. Specifically, our patient carried a small segmental isodisomy of maternal origin affecting 1 Mb of chromosome 4.

6.
BMC Microbiol ; 18(1): 175, 2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30466389

RESUMO

BACKGROUND: The antimicrobial resistance (AMR) phenotypic properties, multiple drug resistance (MDR) gene profiles, and genes related to potential virulence and pathogenic properties of five Enterobacter bugandensis strains isolated from the International Space Station (ISS) were carried out and compared with genomes of three clinical strains. Whole genome sequences of ISS strains were characterized using the hybrid de novo assembly of Nanopore and Illumina reads. In addition to traditional microbial taxonomic approaches, multilocus sequence typing (MLST) analysis was performed to classify the phylogenetic lineage. Agar diffusion discs assay was performed to test antibiotics susceptibility. The draft genomes after assembly and scaffolding were annotated with the Rapid Annotations using Subsystems Technology and RNAmmer servers for downstream analysis. RESULTS: Molecular phylogeny and whole genome analysis of the ISS strains with all publicly available Enterobacter genomes revealed that ISS strains were E. bugandensis and similar to the type strain EB-247T and two clinical isolates (153_ECLO and MBRL 1077). Comparative genomic analyses of all eight E. bungandensis strains showed, a total of 4733 genes were associated with carbohydrate metabolism (635 genes), amino acid and derivatives (496 genes), protein metabolism (291 genes), cofactors, vitamins, prosthetic groups, pigments (275 genes), membrane transport (247 genes), and RNA metabolism (239 genes). In addition, 112 genes identified in the ISS strains were involved in virulence, disease, and defense. Genes associated with resistance to antibiotics and toxic compounds, including the MDR tripartite system were also identified in the ISS strains. A multiple antibiotic resistance (MAR) locus or MAR operon encoding MarA, MarB, MarC, and MarR, which regulate more than 60 genes, including upregulation of drug efflux systems that have been reported in Escherichia coli K12, was also observed in the ISS strains. CONCLUSION: Given the MDR results for these ISS Enterobacter genomes and increased chance of pathogenicity (PathogenFinder algorithm with > 79% probability), these species pose important health considerations for future missions. Thorough genomic characterization of the strains isolated from ISS can help to understand the pathogenic potential, and inform future missions, but analyzing them in in-vivo systems is required to discern the influence of microgravity on their pathogenicity.

7.
Gates Open Res ; 2: 3, 2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-29630066

RESUMO

The Microbe Directory is a collective research effort to profile and annotate more than 7,500 unique microbial species from the MetaPhlAn2 database that includes bacteria, archaea, viruses, fungi, and protozoa. By collecting and summarizing data on various microbes' characteristics, the project comprises a database that can be used downstream of large-scale metagenomic taxonomic analyses, allowing one to interpret and explore their taxonomic classifications to have a deeper understanding of the microbial ecosystem they are studying. Such characteristics include, but are not limited to: optimal pH, optimal temperature, Gram stain, biofilm-formation, spore-formation, antimicrobial resistance, and COGEM class risk rating. The database has been manually curated by trained student-researchers from Weill Cornell Medicine and CUNY-Hunter College, and its analysis remains an ongoing effort with open-source capabilities so others can contribute. Available in SQL, JSON, and CSV (i.e. Excel) formats, the Microbe Directory can be queried for the aforementioned parameters by a microorganism's taxonomy. In addition to the raw database, The Microbe Directory has an online counterpart ( https://microbe.directory/) that provides a user-friendly interface for storage, retrieval, and analysis into which other microbial database projects could be incorporated. The Microbe Directory was primarily designed to serve as a resource for researchers conducting metagenomic analyses, but its online web interface should also prove useful to any individual who wishes to learn more about any particular microbe.

8.
BMC Genomics ; 18(1): 859, 2017 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-29126393

RESUMO

BACKGROUND: Pseudomonas putida is a Gram-negative, non-fermenting bacterium frequently encountered in various environmental niches. P. putida rarely causes disease in humans, though serious infections and outbreaks have been reported from time to time. Some have suggested that P. putida functions as an exchange platform for antibiotic resistance genes (ARG), and thus represents a serious concern in the spread of ARGs to more pathogenic organisms within a hospital. Though poorly understood, the frequency of ARG exchange between P. putida and the more virulent Pseudomonas aeruginosa and its clinical relevance are particularly important for designing efficient infection control strategies, such as deciding whether high-risk patients colonized with a multidrug resistant but typically low pathogenic P. putida strain should be contact isolated or not. RESULTS: In this study, 21,373 screening samples (stool, rectal and throat swab) were examined to determine the presence of P. putida in a high-risk group of haemato-oncology patients during a 28-month period. A total of 89 P. putida group strains were isolated from 85 patients, with 41 of 89 (46.1%) strains harbouring the metallo-beta-lactamase gene bla VIM. These 41 clinical isolates, plus 18 bla VIM positive environmental P. putida isolates, and 17 bla VIM positive P. aeruginosa isolates, were characterized by whole genome sequencing (WGS). We constructed a maximum-likelihood tree to separate the 59 bla VIM positive P. putida group strains into eight distinct phylogenetic clusters. Bla VIM-1 was present in 6 clusters while bla VIM-2 was detected in 4 clusters. Five P. putida group strains contained both, bla VIM-1 and bla VIM-2 genes. In contrast, all P. aeruginosa strains belonged to a single genetic cluster and contained the same ARGs. Apart from bla VIM-2 and sul genes, no other ARGs were shared between P. aeruginosa and P. putida. Furthermore, the bla VIM-2 gene in P. aeruginosa was predicted to be only chromosomally located. CONCLUSION: These data provide evidence that no exchange of comprehensive ARG harbouring mobile genetic elements had occurred between P. aeruginosa and P. putida group strains during the study period, thus eliminating the need to implement enhanced infection control measures for high-risk patients colonized with a bla VIM positiv P. putida group strains in our clinical setting.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Meio Ambiente , Transferência Genética Horizontal , Genômica , Pseudomonas aeruginosa/genética , Pseudomonas putida/genética , Humanos , Filogenia , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/fisiologia
9.
J Biomol Tech ; 28(1): 46-55, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28344519

RESUMO

Amplification of minute quantities of DNA is a fundamental challenge in low-biomass metagenomic and microbiome studies because of potential biases in coverage, guanine-cytosine (GC) content, and altered species abundances. Whole genome amplification (WGA), although widely used, is notorious for introducing artifact sequences, either by amplifying laboratory contaminants or by nonrandom amplification of a sample's DNA. In this study, we investigate the effect of REPLI-g multiple displacement amplification (MDA; Qiagen, Valencia, CA, USA) on sequencing data quality and species abundance detection in 8 paired metagenomic samples and 1 titrated, mixed control sample. We extracted and sequenced genomic DNA (gDNA) from 8 environmental samples and compared the quality of the sequencing data for the MDA and their corresponding non-MDA samples. The degree of REPLI-g MDA bias was evaluated by sequence metrics, species composition, and cross-validating observed species abundance and species diversity estimates using the One Codex and MetaPhlAn taxonomic classification tools. Here, we provide evidence of the overall efficacy of REPLI-g MDA on retaining sequencing data quality and species abundance measurements while providing increased yields of high-fidelity DNA. We find that species abundance estimates are largely consistent across samples, even with REPLI-g amplification, as demonstrated by the Spearman's rank order coefficient (R2 > 0.8). However, REPLI-g MDA often produced fewer classified reads at the species, genera, and family level, resulting in decreased species diversity. We also observed some areas with the PCR "jackpot effect," with varying input DNA values for the Metagenomics Research Group (MGRG) controls at specific genomic loci. We visualize this effect in whole genome coverage plots and with sequence composition analyses and note these caveats of the MDA method. Despite overall concordance of species abundance between the amplified and unamplified samples, these results demonstrate that amplification of DNA using the REPLI-g method has some limitations. These concerns could be addressed by future improvements in the enzymes or methods for REPLI-g to be considered a >99% robust method for increasing the amount of high-fidelity DNA from low-biomass samples or at the very least, accounted for during computational analysis of MDA samples.


Assuntos
Microbiologia Ambiental , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Composição de Bases , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Genoma Bacteriano , Metagenômica , Microbiota/genética
10.
J Biomol Tech ; 28(1): 31-39, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28337070

RESUMO

The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the "Door to Hell" crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.


Assuntos
Microbiologia Ambiental , Microbiota/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Ambientes Extremos , Metagenoma , Tipagem Molecular/normas , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Padrões de Referência , Análise de Sequência de DNA/normas
11.
Proc Natl Acad Sci U S A ; 113(28): E4052-60, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27354520

RESUMO

Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising ∼3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.


Assuntos
Arabidopsis/genética , Inversão Cromossômica , Cromossomos de Plantas , Variação Estrutural do Genoma , Translocação Genética , Dosagem de Genes , Genoma de Planta , Haplótipos , Cariotipagem
13.
Mol Cell ; 57(3): 506-20, 2015 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-25620562

RESUMO

DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the kinase is recruited preferentially to promoters of genes actively transcribed by RNA polymerase II (RNAPII), which are functionally associated with translation, RNA processing, and cell cycle. DYRK1A-bound promoter sequences are highly enriched in a conserved palindromic motif, which is necessary to drive DYRK1A-dependent transcriptional activation. DYRK1A phosphorylates the C-terminal domain (CTD) of RNAPII at Ser2 and Ser5. Depletion of DYRK1A results in reduced association of RNAPII at the target promoters as well as hypophosphorylation of the RNAPII CTD along the target gene bodies. These results are consistent with DYRK1A being a transcriptional regulator by acting as a CTD kinase.


Assuntos
Regiões Promotoras Genéticas , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/genética , Células HeLa , Humanos , Sequências Repetidas Invertidas , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , RNA Polimerase II , Serina/metabolismo , Transcrição Genética
14.
J Antimicrob Chemother ; 70(5): 1322-30, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25583750

RESUMO

OBJECTIVES: Here we report on a long-term outbreak from 2009 to 2012 with an XDR Pseudomonas aeruginosa on two wards at a university hospital in southern Germany. METHODS: Whole-genome sequencing was performed on the outbreak isolates and a core genome was constructed for molecular epidemiological analysis. We applied a time-place-sequence algorithm to improve estimation of transmission probabilities. RESULTS: By using conventional infection control methods we identified 49 P. aeruginosa strains, including eight environmental isolates that belonged to ST308 (by MLST) and carried the metallo-ß-lactamase IMP-8. Phylogenetic analysis on the basis of a non-recombinant core genome that contained 22 outbreak-specific SNPs revealed a pattern of four dominant clades with a strong phylogeographic structure and allowed us to determine the potential temporal origin of the outbreak to July 2008, 1 year before the index case was diagnosed. Superspreaders at the root of clades exhibited a high number of probable and predicted transmissions, indicating their exceptional position in the outbreak. CONCLUSIONS: Our results suggest that the initial expansion of dominant sublineages was driven by a few superspreaders, while environmental contamination seemed to sustain the outbreak for a long period despite regular environmental control measures.


Assuntos
Surtos de Doenças , Farmacorresistência Bacteriana Múltipla , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , Transmissão de Doença Infecciosa , Microbiologia Ambiental , Estudos Epidemiológicos , Genoma Bacteriano , Alemanha/epidemiologia , Hospitais Universitários , Humanos , Epidemiologia Molecular , Tipagem Molecular , Infecções por Pseudomonas/transmissão , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Análise de Sequência de DNA , Análise Espaço-Temporal
15.
Genome Biol ; 15(4): R57, 2014 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-24708865

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are established regulators of development, cell identity and disease. Although nearly two thousand human miRNA genes are known and new ones are continuously discovered, no attempt has been made to gauge the total miRNA content of the human genome. RESULTS: Employing an innovative computational method on massively pooled small RNA sequencing data, we report 2,469 novel human miRNA candidates of which 1,098 are validated by in-house and published experiments. Almost 300 candidates are robustly expressed in a neuronal cell system and are regulated during differentiation or when biogenesis factors Dicer, Drosha, DGCR8 or Ago2 are silenced. To improve expression profiling, we devised a quantitative miRNA capture system. In a kidney cell system, 400 candidates interact with DGCR8 at transcript positions that suggest miRNA hairpin recognition, and 1,000 of the new miRNA candidates interact with Ago1 or Ago2, indicating that they are directly bound by miRNA effector proteins. From kidney cell CLASH experiments, in which miRNA-target pairs are ligated and sequenced, we observe hundreds of interactions between novel miRNAs and mRNA targets. The novel miRNA candidates are specifically but lowly expressed, raising the possibility that not all may be functional. Interestingly, the majority are evolutionarily young and overrepresented in the human brain. CONCLUSIONS: In summary, we present evidence that the complement of human miRNA genes is substantially larger than anticipated, and that more are likely to be discovered in the future as more tissues and experimental conditions are sequenced to greater depth.


Assuntos
Genoma Humano , MicroRNAs/genética , Proteínas Argonauta/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Células MCF-7 , MicroRNAs/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo
16.
EMBO J ; 31(15): 3323-33, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22781127

RESUMO

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, our understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, we have identified binding regions for the Hox TF Deformed (Dfd) in the Drosophila genome. Our analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. We also find that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, our results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo.


Assuntos
Drosophila/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Drosophila/embriologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Genes de Insetos , Código das Histonas/genética , Código das Histonas/fisiologia , Proteínas de Homeodomínio/metabolismo , Modelos Biológicos , Ligação Proteica , Fatores de Transcrição/fisiologia , Ativação Transcricional
17.
PLoS Genet ; 8(3): e1002582, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22438831

RESUMO

Apoptosis is essential to prevent oncogenic transformation by triggering self-destruction of harmful cells, including those unable to differentiate. However, the mechanisms linking impaired cell differentiation and apoptosis during development and disease are not well understood. Here we report that the Drosophila transcription factor Cut coordinately controls differentiation and repression of apoptosis via direct regulation of the pro-apoptotic gene reaper. We also demonstrate that this regulatory circuit acts in diverse cell lineages to remove uncommitted precursor cells in status nascendi and thereby interferes with their potential to develop into cancer cells. Consistent with the role of Cut homologues in controlling cell death in vertebrates, we find repression of apoptosis regulators by Cux1 in human cancer cells. Finally, we present evidence that suggests that other lineage-restricted specification factors employ a similar mechanism to put the brakes on the oncogenic process.


Assuntos
Apoptose , Diferenciação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Linhagem da Célula , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores de Tumor , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo
18.
Curr Opin Biotechnol ; 22(4): 590-4, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21497080

RESUMO

Adaptive laboratory evolution (ALE) strategies allow for the metabolic engineering of microorganisms by combining genetic variation with the selection of beneficial mutations in an unbiased fashion. These ALE strategies have been proven highly effective in the optimization of production strains. In contrast to rational engineering strategies and directed modification of specific enzymes, ALE has the advantage of letting nonintuitive beneficial mutations occur in many different genes and regulatory regions in parallel. So far, the majority of applications of ALE in metabolic engineering have used well-characterized platform organisms such as Saccharomyces cerevisiae and Escherichia coli; however, applications for other microorganisms are on the rise. This review will focus on current applications of ALE as a tool for metabolic engineering and discuss advancements and achievements that have been made in this field.


Assuntos
Evolução Molecular Direcionada , Escherichia coli/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Redes e Vias Metabólicas , Saccharomyces cerevisiae/genética
19.
PLoS Genet ; 5(3): e1000412, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19282966

RESUMO

Hox proteins play fundamental roles in controlling morphogenetic diversity along the anterior-posterior body axis of animals by regulating distinct sets of target genes. Within their rather broad expression domains, individual Hox proteins control cell diversification and pattern formation and consequently target gene expression in a highly localized manner, sometimes even only in a single cell. To achieve this high-regulatory specificity, it has been postulated that Hox proteins co-operate with other transcription factors to activate or repress their target genes in a highly context-specific manner in vivo. However, only a few of these factors have been identified. Here, we analyze the regulation of the cell death gene reaper (rpr) by the Hox protein Deformed (Dfd) and suggest that local activation of rpr expression in the anterior part of the maxillary segment is achieved through a combinatorial interaction of Dfd with at least eight functionally diverse transcriptional regulators on a minimal enhancer. It follows that context-dependent combinations of Hox proteins and other transcription factors on small, modular Hox response elements (HREs) could be responsible for the proper spatio-temporal expression of Hox targets. Thus, a large number of transcription factors are likely to be directly involved in Hox target gene regulation in vivo.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/fisiologia , Elementos de Resposta/genética , Fatores de Transcrição/genética , Animais , Drosophila melanogaster/genética
20.
Development ; 134(2): 381-92, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17166915

RESUMO

Functional diversification of body parts is dependent on the formation of specialized structures along the various body axes. In animals, region-specific morphogenesis along the anteroposterior axis is controlled by a group of conserved transcription factors encoded by the Hox genes. Although it has long been assumed that Hox proteins carry out their function by regulating distinct sets of downstream genes, only a small number of such genes have been found, with very few having direct roles in controlling cellular behavior. We have quantitatively identified hundreds of Hox downstream genes in Drosophila by microarray analysis, and validated many of them by in situ hybridizations on loss- and gain-of-function mutants. One important finding is that Hox proteins, despite their similar DNA-binding properties in vitro, have highly specific effects on the transcriptome in vivo, because expression of many downstream genes respond primarily to a single Hox protein. In addition, a large fraction of downstream genes encodes realizator functions, which directly affect morphogenetic processes, such as orientation and rate of cell divisions, cell-cell adhesion and communication, cell shape and migration, or cell death. Focusing on these realizators, we provide a framework for the morphogenesis of the maxillary segment. As the genomic organization of Hox genes and the interaction of Hox proteins with specific co-factors are conserved in vertebrates and invertebrates, and similar classes of downstream genes are regulated by Hox proteins across the metazoan phylogeny, our findings represent a first step toward a mechanistic understanding of morphological diversification within a species as well as between species.


Assuntos
Drosophila/embriologia , Drosophila/genética , Genes Homeobox , Genes de Insetos , Animais , Padronização Corporal/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Maxila/embriologia , Morfogênese , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade da Espécie
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